Methods and materials for assessing loss of heterozygosity

ABSTRACT

This document provides methods and materials involved in assessing samples (e.g., cancer cells) for the presence of a loss of heterozygosity (LOH) signature. For example, methods and materials for determining whether or not a cell (e.g., a cancer cell) contains an LOH signature are provided. Materials and methods for identifying cells (e.g., cancer cells) having a deficiency in homology directed repair (HDR) as well as materials and methods for identifying cancer patients likely to respond to a particular cancer treatment regimen also are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. Pat.Application Serial No. 15/192,497, filed Jun. 24, 2016, which is acontinuation of U.S. Pat. Application Serial No. 14/307,708 filed Jun.18, 2014, now U.S. Pat. No. 9,388,472, which is a continuation ofInternational Application Serial No. PCT/US2012/071380, filed Dec. 31,2012, which claims the benefit of priority to U.S. ProvisionalApplication Serial No. 61/578,713 filed Dec. 21, 2011 and to U.S.Provisional Pat. Application Serial No. 61/654,402 filed Jun. 1, 2012,the entire contents of each of which are hereby incorporated byreference.

BACKGROUND 1. Technical Field

This document relates to methods and materials involved in assessingsamples (e.g., cancer cells) for the presence of a loss ofheterozygosity (LOH) signature. For example, this document providesmethods and materials for determining whether or not a cell (e.g., acancer cell) contains an LOH signature. This document also providesmaterials and methods for identifying cells (e.g., cancer cells) havinga deficiency in homology directed repair (HDR) as well as materials andmethods for identifying cancer patients likely to respond to aparticular cancer treatment regimen. Throughout this document, unlessindicated otherwise, HDR deficiency and HRD (homologous repairdeficiency) are used synonymously.

2. Background Information

Cancer is a serious public health problem, with 562,340 people in theUnited States of America dying of cancer in 2009 alone. American CancerSociety, Cancer Facts & Figures 2009 (available at American CancerSociety website). One of the primary challenges in cancer treatment isdiscovering relevant, clinically useful characteristics of a patient’sown cancer and then, based on these characteristics, administering atreatment plan best suited to the patient’s cancer. While strides havebeen made in this field of personalized medicine, there is still asignificant need for better molecular diagnostic tools to characterizepatients’ cancers.

SUMMARY

In general, one aspect of this invention features a method for assessingLOH in a cancer cell or genomic DNA thereof. In some embodiments, themethod comprises, or consists essentially of, (a) detecting, in a cancercell or genomic DNA derived therefrom, LOH regions in at least one pairof human chromosomes of the cancer cell (e.g., any pair of humanchromosomes other than a human X/Y sex chromosome pair); and (b)determining the number and size (e.g., length) of said LOH regions. Insome embodiments, LOH regions are analyzed in a number of chromosomepairs that are representative of the entire genome (e.g., enoughchromosomes are analyzed such that the number and size of LOH regionsare expected to be representative of the number and size of LOH regionsacross the genome). In some embodiments, the method further comprisesdetermining the total number of LOH regions that are longer than about1.5, 5, 12, 13, 14, 15, 16, 17 or more (preferably 14, 15, 16 or more,more preferably 15 or more) megabases but shorter than the entire lengthof the respective chromosome which the LOH region is located within(Indicator LOH Regions). Alternatively or additionally, the totalcombined length of such Indicator LOH Regions is determined. In somespecific embodiments, if that total number of Indicator LOH Regions ortotal combined length of Indicator LOH Regions is equal to or greaterthan a predetermined reference number, then said cancer cell or genomicDNA or a patient having said cancer cell or genomic DNA is identified ashaving an HDR-deficiency LOH signature.

An alternative method for assessing LOH in a cancer cell or genomic DNAthereof is also provided which comprises, or consists essentially of,(a) detecting, in a cancer cell or genomic DNA derived therefrom, LOHregions in at least one pair of human chromosomes of the cancer cell,wherein the at least one pair of human chromosomes is not a human X/Ysex chromosome pair; and (b) determining the total number and/orcombined length of LOH regions, in the at least one pair of humanchromosomes, that are longer than a first length but shorter than thelength of the whole chromosome containing the LOH region, wherein thefirst length is about 1.5 or more (or 5, 10, 13, 14, 15, 16 or more,preferably 15 or more) megabases. In some specific embodiments, if thattotal number or combined length is equal to or greater than apredetermined reference number, then said cancer cell or genomic DNA ora patient having said cancer cell or genomic DNA is identified as havingan HDR-deficiency LOH signature.

In another aspect, the present invention provides a method of predictingthe status of BRCA1 and BRCA2 genes in a cancer cell. The methodcomprises, or consists essentially of, determining, in the cancer cell,the total number and/or combined length of LOH regions in at least onepair of human chromosomes of the cancer cell that are longer than afirst length but shorter than the length of the whole chromosomecontaining the LOH region, wherein the at least one pair of humanchromosomes is not a human X/Y sex chromosome pair, wherein the firstlength is about 1.5 or more (or 5, 10 or more, preferably about 15 ormore) megabases; and correlating the total number or combined lengththat is greater than a reference number with an increased likelihood ofa deficiency in the BRCA1 or BRCA2 gene.

In another aspect, this invention provides a method of predicting thestatus of HDR in a cancer cell. The method comprises, or consistsessentially of, determining, in the cancer cell, the total number and/orcombined length of LOH regions in at least one pair of human chromosomesof the cancer cell that are longer than a first length but shorter thanthe length of the whole chromosome containing the LOH region, whereinthe at least one pair of human chromosomes is not a human X/Y sexchromosome pair, wherein the first length is about 1.5 or more (or 5, 10or more, preferably about 15 or more) megabases; and correlating thetotal number or combined length that is greater than a reference numberwith an increased likelihood of a deficiency in HDR.

In another aspect, this invention provides a method of predicting acancer patient’s response to a cancer treatment regimen comprising a DNAdamaging agent, an anthracycline, a topoisomerase I inhibitor,radiation, and/or a PARP inhibitor. The method comprises, or consistsessentially of, determining, in a cancer cell from the cancer patient,the number and/or combined length of LOH regions in at least one pair ofhuman chromosomes of a cancer cell of the cancer patient that are longerthan a first length but shorter than the length of the whole chromosomecontaining the LOH region, wherein the at least one pair of humanchromosomes is not a human X/Y sex chromosome pair, wherein the firstlength is about 1.5 or more (or 5, 10 or more, preferably about 15 ormore) megabases; and correlating the total number or combined lengththat is greater than a reference number with an increased likelihoodthat the cancer patient will respond to the cancer treatment regimen. Insome embodiments, the patients are treatment naïve patients.

In another aspect, present invention relates to a method of predicting acancer patient’s response to a treatment regimen. The method comprises,or consists essentially of, determining, in a cancer cell from thecancer patient, the total number and/or combined length of LOH regionsin at least one pair of human chromosomes of a cancer cell of the cancerpatient that are longer than a first length but shorter than the lengthof the whole chromosome containing the LOH region, wherein the at leastone pair of human chromosomes is not a human X/Y sex chromosome pair,wherein the first length is about 1.5 or more (or 5, 10 or more,preferably about 15 or more) megabases; and correlating the total numberor combined length that is greater than a reference number with anincreased likelihood that the cancer patient will not respond to atreatment regimen including paclitaxel or docetaxel.

In another aspect, this invention is directed to a method of treatingcancer. The method comprises, or consists essentially of, (a)determining, in a cancer cell from a cancer patient or genomic DNAobtained therefrom, the total number and/or combined length of LOHregions in at least one pair of human chromosomes of the cancer cellthat are longer than a first length but shorter than the length of thewhole chromosome containing the LOH region, wherein the at least onepair of human chromosomes is not a human X/Y sex chromosome pair,wherein the first length is about 1.5 or more (or 5, 10 or more,preferably about 15 or more) megabases; and (b) administering to thecancer patient a cancer treatment regimen comprising one or more drugschosen from the group consisting of DNA damaging agents, anthracyclines,topoisomerase I inhibitors, and PARP inhibitors, if the total number orcombined length of LOH regions is greater than a reference number. Insome embodiments, the patients are treatment naïve patients.

In some embodiments of any one or more of the methods described in thepreceding six paragraphs, any one or more of the following can beapplied as appropriate. The LOH regions can be determined in at leasttwo, five, ten, or 21 pairs of human chromosomes. The cancer cell can bean ovarian, breast, or esophageal cancer cell. The first length can beabout 6, 12, or about 15 or more megabases. The reference number can be6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 20 or greater. The atleast one pair of human chromosomes can exclude human chromosome 17. TheDNA damaging agent can be cisplatin, carboplatin, oxalaplatin, orpicoplatin, the anthracycline can be epirubincin or doxorubicin, thetopoisomerase I inhibitor can be campothecin, topotecan, or irinotecan,or the PARP inhibitor can be iniparib, olaparib or velapirib.

In another aspect, this invention features the use of one or more drugsselected from the group consisting of DNA damaging agents,anthracyclines, topoisomerase I inhibitors, and PARP inhibitors, in themanufacture of a medicament useful for treating a cancer in a patientidentified as having a cancer cell determined to have a total of 5, 8,9, 10, 12, 15, 17, 20 or more Indicator LOH Regions. The Indicator LOHRegions can be determined in at least two, five, ten, or 21 pairs ofhuman chromosomes. The cancer cell can be an ovarian, breast, oresophageal cancer cell. The Indicator LOH Regions can have a length ofabout 6, 12, or 15 or more megabases. The Indicator LOH Regions can bepresent on a chromosome other than human chromosome 17. The DNA damagingagent can be a platinum-based chemotherapy drug, the anthracycline canbe epirubincin or doxorubicin, the topoisomerase I inhibitor can becampothecin, topotecan, or irinotecan, or the PARP inhibitor can beiniparib, olaparib or velapirib. In some embodiments, the patients aretreatment naïve patients.

In another aspect, this invention features the use of a plurality ofoligonucleotides capable of hybridizing to a plurality of polymorphicregions of human genomic DNA, in the manufacture of a diagnostic kituseful for determining the total number or combined length of IndicatorLOH Regions in at least a chromosome pair of a human cancer cellobtained from a cancer patient, and for detecting (a) an increasedlikelihood of a deficiency in the BRCA1 or BRCA2 gene in the cancercell, (b) an increased likelihood of a deficiency in HDR in the cancercell, or (c) an increased likelihood that the cancer patient willrespond to cancer treatment regimen comprising a DNA damaging agent, ananthracycline, a topoisomerase I inhibitor, radiation, or a PARPinhibitor. The Indicator LOH Regions can be determined in at least two,five, ten, or 21 pairs of human chromosomes. The cancer cell can be anovarian, breast, or esophageal cancer cell. The Indicator LOH Regionscan have a length of about 6, 12, or 15 or more megabases. The IndicatorLOH Regions can be present on a chromosome other than human chromosome17.

In another aspect, this invention features a system for determining LOHstatus of a cancer cell of a cancer patient. The system comprises, orconsists essentially of, (a) a sample analyzer configured to produce aplurality of signals about genomic DNA of at least one pair of humanchromosomes of the cancer cell, and (b) a computer sub-system programmedto calculate, based on the plurality of signals, the number or combinedlength of Indicator LOH Regions in the at least one pair of humanchromosomes. The computer sub-system can be programmed to compare thenumber or combined length of Indicator LOH Regions to a reference numberto determine (a) a likelihood of a deficiency in BRCA1 and/or BRCA2genes in the cancer cell, (b) a likelihood of a deficiency in HDR in thecancer cell, or (c) a likelihood that the cancer patient will respond tocancer treatment regimen comprising a DNA damaging agent, ananthracycline, a topoisomerase I inhibitor, radiation, or a PARPinhibitor. The system can comprise an output module configured todisplay the likelihood of (a), (b), or (c). The system can comprise anoutput module configured to display a recommendation for the use of thecancer treatment regimen. The Indicator LOH Regions can be determined inat least two, five, ten, or 21 pairs of human chromosomes. The cancercell can be an ovarian, breast, or esophageal cancer cell. The IndicatorLOH Regions can have a length of about 6, 12, or 15 or more megabases.The Indicator LOH Regions can be present on chromosomes other than ahuman chromosome 17. The DNA damaging agent can be a platinum-basedchemotherapy drug, the anthracycline can be epirubincin or doxorubicin,the topoisomerase I inhibitor can be campothecin, topotecan, oririnotecan, or the PARP inhibitor can be iniparib, olaparib orvelapirib.

In another aspect, the invention provides a computer program productembodied in a computer readable medium that, when executing on acomputer, provides instructions for detecting the presence or absence ofany LOH region along one or more of human chromosomes other than thehuman X and Y sex chromosomes, and the LOH region having a length ofabout 1.5 or more (or 5, 10 or more, preferably 15 or more) megabasesbut shorter than the length of the whole chromosome containing the LOHregion; and determining the total number or combined length of the LOHregions in the one or more chromosome pairs. The computer programproduct can include other instructions. The Indicator LOH Regions can bedetermined in at least two, five, ten or 21 pairs of human chromosomes.The cancer cell can be an ovarian, breast, or esophageal cancer cell.The Indicator LOH Regions can have a length of about 6, 12, or 15 ormore megabases. The Indicator LOH Regions can be present on chromosomesother than a human chromosome 17. The DNA damaging agent can be aplatinum-based chemotherapy drug, the anthracycline can be epirubincinor doxorubicin, the topoisomerase I inhibitor can be campothecin,topotecan, or irinotecan, or the PARP inhibitor can be iniparib,olaparib or velapirib.

In another aspect, the present invention provides a diagnostic kit. Thekit comprises, or consists essentially of, at least 500 oligonucleotidescapable of hybridizing to a plurality of polymorphic regions of humangenomic DNA; and a computer program product provided herein. Thecomputer program product can be embodied in a computer readable mediumthat, when executing on a computer, provides instructions for detectingthe presence or absence of any LOH region along one or more of humanchromosomes other than the human X and Y sex chromosomes, and the LOHregion having a length of about 1.5 or more (or 5 or 10 or more,preferably about 15 or more) megabases but shorter than the length ofthe whole chromosome containing the LOH region; and determining thetotal number and/or combined length of the LOH region in the one or morechromosome pairs.

In another aspect, this document features a method for assessing cancercells of a patient for the presence of an LOH signature. The methodcomprises, or consists essentially of, (a) detecting the presence ofmore than a reference number of LOH regions in at least one pair ofhuman chromosomes of a cancer cell of the cancer patient that are longerthan a first length but shorter than the length of the whole chromosomecontaining the LOH region, wherein the at least one pair of humanchromosomes is not a human X/Y sex chromosome pair, wherein the firstlength is about 1.5 or more megabases, and (b) identifying the patientas having cancer cells with the LOH signature.

In another aspect, this document features a method for assessing cancercells of a patient for the presence of an HDR deficient status. Themethod comprises, or consists essentially of, (a) detecting the presenceof more than a reference number of LOH regions in at least one pair ofhuman chromosomes of a cancer cell of the cancer patient that are longerthan a first length but shorter than the length of the whole chromosomecontaining the LOH region, wherein the at least one pair of humanchromosomes is not a human X/Y sex chromosome pair, wherein the firstlength is about 1.5 or more megabases, and (b) identifying the patientas having cancer cells with the HDR deficient status.

In another aspect, this document features a method for assessing cancercells of a patient for the presence of a genetic mutation within a genefrom an HDR pathway. The method comprises, or consists essentially of,(a) detecting the presence of more than a reference number of LOHregions in at least one pair of human chromosomes of a cancer cell ofthe cancer patient that are longer than a first length but shorter thanthe length of the whole chromosome containing the LOH region, whereinthe at least one pair of human chromosomes is not a human X/Y sexchromosome pair, wherein the first length is about 1.5 or moremegabases, and (b) identifying the patient as having cancer cells withthe genetic mutation.

In another aspect, this document features a method for determining if apatient is likely to respond to a cancer treatment regimen comprisingadministering radiation or a drug selected from the group consisting ofDNA damaging agents, anthracyclines, topoisomerase I inhibitors, andPARP inhibitors. The method comprises, or consists essentially of, (a)detecting the presence of more than a reference number of LOH regions inat least one pair of human chromosomes of a cancer cell of the cancerpatient that are longer than a first length but shorter than the lengthof the whole chromosome containing the LOH region, wherein the at leastone pair of human chromosomes is not a human X/Y sex chromosome pair,wherein the first length is about 1.5 or more megabases, and (b)identifying the patient as being likely to respond to the cancertreatment regimen.

In another aspect, this document features a method for assessing apatient. The method comprises, or consists essentially of, (a)determining that the patient comprises cancer cells having an LOHsignature, wherein the presence of more than a reference number of LOHregions in at least one pair of human chromosomes of a cancer cell ofthe cancer patient that are longer than a first length but shorter thanthe length of the whole chromosome containing the LOH region indicatesthat the cancer cells have the LOH signature, wherein the at least onepair of human chromosomes is not a human X/Y sex chromosome pair,wherein the first length is about 1.5 or more megabases, and (b)diagnosing the patient as having cancer cells with the LOH signature.

In another aspect, this document features a method for assessing apatient. The method comprises, or consists essentially of, (a)determining that the patient comprises cancer cells having an HDRdeficiency status, wherein the presence of more than a reference numberof LOH regions in at least one pair of human chromosomes of a cancercell of the cancer patient that are longer than a first length butshorter than the length of the whole chromosome containing the LOHregion indicates that the cancer cells have the HDR deficiency status,wherein the at least one pair of human chromosomes is not a human X/Ysex chromosome pair, wherein the first length is about 1.5 or moremegabases, and (b) diagnosing the patient as having cancer cells withthe HDR deficient status.

In another aspect, this document features a method for assessing apatient. The method comprises, or consists essentially of, (a)determining that the patient comprises cancer cells having a geneticmutation within a gene from an HDR pathway, wherein the presence of morethan a reference number of LOH regions in at least one pair of humanchromosomes of a cancer cell of the cancer patient that are longer thana first length but shorter than the length of the whole chromosomecontaining the LOH region indicates that the cancer cells have thegenetic mutation, wherein the at least one pair of human chromosomes isnot a human X/Y sex chromosome pair, wherein the first length is about1.5 or more megabases, and (b) diagnosing the patient as having cancercells with the genetic mutation.

In another aspect, this document features a method for assessing apatient for a likelihood to respond to a cancer treatment regimencomprising administering radiation or a drug selected from the groupconsisting of DNA damaging agents, anthracyclines, topoisomerase Iinhibitors, and PARP inhibitors. The method comprises, or consistsessentially of, (a) determining that the patient comprises cancer cellshaving an LOH signature, wherein the presence of more than a referencenumber of LOH regions in at least one pair of human chromosomes of acancer cell of the cancer patient that are longer than a first lengthbut shorter than the length of the whole chromosome containing the LOHregion indicates that the cancer cells have the LOH signature, whereinthe at least one pair of human chromosomes is not a human X/Y sexchromosome pair, wherein the first length is about 1.5 or moremegabases, and (b) diagnosing, based at least in part on the presence ofthe LOH signature, the patient as being likely to respond to the cancertreatment regimen.

In another aspect, this document features a method for performing adiagnostic analysis of a cancer cell of a patient. The method comprises,or consists essentially of, (a) detecting the presence of more than areference number of LOH regions in at least one pair of humanchromosomes of the cancer cell that are longer than a first length butshorter than the length of the whole chromosome containing the LOHregion, wherein the at least one pair of human chromosomes is not ahuman X/Y sex chromosome pair, wherein the first length is about 1.5 ormore megabases, and (b) identifying the patient as having cancer cellswith an LOH signature.

In another aspect, this document features a method for performing adiagnostic analysis of a cancer cell of a patient. The method comprises,or consists essentially of, (a) detecting the presence of more than areference number of LOH regions in at least one pair of humanchromosomes of the cancer cell that are longer than a first length butshorter than the length of the whole chromosome containing the LOHregion, wherein the at least one pair of human chromosomes is not ahuman X/Y sex chromosome pair, wherein the first length is about 1.5 ormore megabases, and (b) identifying the patient as having cancer cellswith a HDR deficient status.

In another aspect, this document features a method for performing adiagnostic analysis of a cancer cell of a patient. The method comprises,or consists essentially of, (a) detecting the presence of more than areference number of LOH regions in at least one pair of humanchromosomes of the cancer cell that are longer than a first length butshorter than the length of the whole chromosome containing the LOHregion, wherein the at least one pair of human chromosomes is not ahuman X/Y sex chromosome pair, wherein the first length is about 1.5 ormore megabases, and (b) identifying the patient as having cancer cellswith a genetic mutation within a gene from an HDR pathway.

In another aspect, this document features a method for performing adiagnostic analysis of a cancer cell of a patient to determine if thecancer patient is likely to respond to a cancer treatment regimencomprising administering radiation or a drug selected from the groupconsisting of DNA damaging agents, anthracyclines, topoisomerase Iinhibitors, and PARP inhibitors. The method comprises, or consistsessentially of, (a) detecting the presence of more than a referencenumber of LOH regions in at least one pair of human chromosomes of thecancer cell that are longer than a first length but shorter than thelength of the whole chromosome containing the LOH region, wherein the atleast one pair of human chromosomes is not a human X/Y sex chromosomepair, wherein the first length is about 1.5 or more megabases, and (b)identifying the patient as being likely to respond to the cancertreatment regimen.

In another aspect, this document features a method for diagnosing apatient as having cancer cells having an LOH signature. The methodcomprises, or consists essentially of, (a) determining that the patientcomprises cancer cells having the LOH signature, wherein the presence ofmore than a reference number of LOH regions in at least one pair ofhuman chromosomes of a cancer cell of the cancer patient that are longerthan a first length but shorter than the length of the whole chromosomecontaining the LOH region indicates that the cancer cells have the LOHsignature, wherein the at least one pair of human chromosomes is not ahuman X/Y sex chromosome pair, wherein the first length is about 1.5 ormore megabases, and (b) diagnosing the patient as having cancer cellswith the LOH signature.

In another aspect, this document features a method for diagnosing apatient as having cancer cells with an HDR deficient status. The methodcomprises, or consists essentially of, (a) determining that the patientcomprises cancer cells having the HDR deficiency status, wherein thepresence of more than a reference number of LOH regions in at least onepair of human chromosomes of a cancer cell of the cancer patient thatare longer than a first length but shorter than the length of the wholechromosome containing the LOH region indicates that the cancer cellshave the HDR deficiency status, wherein the at least one pair of humanchromosomes is not a human X/Y sex chromosome pair, wherein the firstlength is about 1.5 or more megabases, and (b) diagnosing the patient ashaving cancer cells with the HDR deficient status.

In another aspect, this document features a method for diagnosing apatient as having cancer cells with a genetic mutation within a genefrom an HDR pathway. The method comprises, or consists essentially of,(a) determining that the patient comprises cancer cells having thegenetic mutation, wherein the presence of more than a reference numberof LOH regions in at least one pair of human chromosomes of a cancercell of the cancer patient that are longer than a first length butshorter than the length of the whole chromosome containing the LOHregion indicates that the cancer cells have the genetic mutation,wherein the at least one pair of human chromosomes is not a human X/Ysex chromosome pair, wherein the first length is about 1.5 or moremegabases, and (b) diagnosing the patient as having cancer cells withthe genetic mutation.

In another aspect, this document features a method for diagnosing apatient as being a candidate for a cancer treatment regimen comprisingadministering radiation or a drug selected from the group consisting ofDNA damaging agents, anthracyclines, topoisomerase I inhibitors, andPARP inhibitors. The method comprises, or consists essentially of, (a)determining that the patient comprises cancer cells having an LOHsignature, wherein the presence of more than a reference number of LOHregions in at least one pair of human chromosomes of a cancer cell ofthe cancer patient that are longer than a first length but shorter thanthe length of the whole chromosome containing the LOH region indicatesthat the cancer cells have the LOH signature, wherein the at least onepair of human chromosomes is not a human X/Y sex chromosome pair,wherein the first length is about 1.5 or more megabases, and (b)diagnosing, based at least in part on the presence of the LOH signature,the patient as being likely to respond to the cancer treatment regimen.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe description and accompanying drawings below. The materials, methods,and examples are illustrative only and not intended to be limiting.Other features, objects, and advantages of the invention will beapparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph plotting allele dosages of breast cancer cells from abreast cancer patient along chromosome 1 as determined using a SNParray. The arrow indicates a transition between a region ofheterozygosity and an LOH region.

FIG. 2 is a graph plotting allele dosages of breast cancer cells for thesame breast cancer patient as on FIG. 1 along chromosome 1 as determinedusing high-throughput sequencing. The arrow indicates a transitionbetween a region of heterozygosity and an LOH region.

FIG. 3 is a flow chart of an example process for assessing the genome ofa cell (e.g., a cancer cell) for an LOH signature.

FIG. 4 is a diagram of an example of a computer device and a mobilecomputer device that can be used to implement the techniques describedherein.

FIG. 5 is a graph plotting the length distribution of LOH regionsdetected in ovarian cancer cells from 62 human patients. The adjustedlength refers to the fraction of chromosomes arms covered by LOHregions.

FIG. 6 is a graph plotting the number of LOH regions longer than 15 Mband shorter than the entire chromosome for a training set of ovariancancer cell samples with intact or deficient BRCA1 and BRCA2 genes. Thesize of the circles is proportional to the number of samples with suchnumber of LOH regions.

FIG. 7 is a graph plotting the number of LOH regions longer than 15 Mband shorter than the entire chromosome for a training and validationsets of ovarian cancer cell samples with intact or deficient BRCA1 andBRCA2 genes. The size of the circles is proportional to the number ofsamples with such number of LOH regions.

FIG. 8 is a graph plotting the number of LOH regions longer than 15 Mband shorter than the entire chromosome for ovarian cancer cell sampleswith somatic BRCA mutations, with germline BRCA mutations, with lowBRCA1 expression, or with intact BRCA (BRCA normal). The size of thecircles is proportional to the number of samples with such number of LOHregions.

FIG. 9 is a table showing the percent of ovarian cancer samples that areBRCA deficient, HDR deficient/BRCA intact, and HDR intact.

FIG. 10 is a graph plotting the number of LOH regions longer than 15 Mband shorter than the entire chromosome for cancer cell lines for theindicated cancers. The size of the circles is proportional to the numberof samples with such number of LOH regions.

FIG. 11 is a graph plotting the number of LOH regions longer than 15 Mband shorter than the entire chromosome for lung cancer samples.

FIG. 12 is a graph plotting the percentage of the indicated cancers orcancer cell lines having an HDR deficiency.

FIG. 13 contains graphs plotting the IC₅₀ values (Log₁₀(IC₅₀) ofcamptothecin, as well as averaged Log₁₀(IC₅₀) values for platinumcompounds (oxaliplatin, cisplatin, and carboplatin), or anthracyclines(doxorubicin and epirubicin) when exposed to 29 breast cancer cell lineshaving the indicated number of LOH regions longer than 15 Mb and shorterthan the entire chromosome or the IC₅₀ values (Log₁₀(IC₅₀)) ofpaclitaxel when exposed to 27 ovarian cancer cell lines having theindicated number of LOH regions longer than 15 Mb and shorter than theentire chromosome. The dashed lines place a threshold number at nine.

FIG. 14 is a labeled version of a graph from FIG. 13 that plots theaveraged Log₁₀(IC₅₀) values of platinum compounds (oxaliplatin,cisplatin, and carboplatin) when exposed to 29 breast cancer cell lineshaving the indicated number of LOH regions longer than 15 Mb and shorterthan the entire chromosome.

FIG. 15 is a flow chart of an example computational process foridentifying LOH loci and regions.

FIG. 16 shows fraction of lengths of LOH regions vs. length of theseregions adjusted on the length of chromosome arm. The largest adjustedvalue on this figure is equal to two corresponding to LOH over theentire chromosome.

FIG. 17A shows HRD score in tumor samples. Circles correspond to BRCA1or BRCA2 deficient samples or BRCA1 and BRCA2 intact samples asindicated. Combined area under circles for each row is the same. Thearea of each individual circle is proportional to the number of sampleswith the corresponding number of LOH regions. HRD score for the firstcohort (46 of 152 samples were BRCA1 or BRCA2 deficient).

FIG. 17B shows HRD score in tumor samples. Circles correspond to BRCA1or BRCA2 deficient sample or BRCA1 and BRCA2 intact samples asindicated. Combined area under circles for each row is the same. Thearea of each individual circle is proportional to the number of sampleswith the corresponding number of LOH regions. HRD score for the secondcohort (19 of 53 samples were BRCA1 or BRCA2 deficient).

FIG. 17C shows HRD score in tumor samples. Circles correspond to BRCA1or BRCA2 deficient samples or BRCA1 and BRCA2 intact samples asindicated. Combined area under circles for each row is the same. Thearea of each individual circle is proportional to the number of sampleswith the corresponding number of LOH regions. HRD score for the thirdcohort (146 of 435 samples were BRCA1 or BRCA2 deficient).

FIG. 17D shows HRD score in tumor samples. Circles correspond to BRCA1or BRCA2 deficient samples or BRCA1 and BRCA2 intact samples asindicated. Combined area under circles for each row is the same. Thearea of each individual circle is proportional to the number of sampleswith the corresponding number of LOH regions. HRD score for the combineddata from all three cohorts. Row A: 224 samples with either BRCA1, orBRCA2, or RAD51C deficient genes; B: 84 BRCA1 mutants; C: 43 BRCA2mutants; D: 82 samples with low expression or methylation of BRCA1; E:13 samples with methylation of RAD51C. Red circles: 416 samples withBRCA1, BRCA2, and RAD51C intact genes.

FIG. 18A shows a comparison of HRD scores in cancer cell lines. Celllines with intact BRCA1 or BRCA2 are indicated: 30 intact non-ovariancell lines; 22 intact ovarian cell lines. 6 carriers of heterozygousmutations in either BRCA1 or BRCA2, 2 carriers of homozygous mutationswith reversion in either BRCA1 or BRCA2, and 7 carriers of homozygousmutations in either BRCA1 or BRCA2 or with methylated BRCA1. Thecombined area under the circles for each row is the same. The area undereach individual circle is proportional to the number of samples with thecorresponding number of LOH regions.

FIG. 18B shows Kaplan-Meier plot of OS post-surgery for HRD score splitat its median. These data were generated using 507 samples from the TCGAdataset for which copy number data and survival information wereavailable. Median OS for samples with high and low HRD score were 1499(95% Cl=(1355-1769)) and 1163 (95% CI=(1081-1354)) days, respectively.

FIG. 19 shows the correlation between LOH scores and HR deficiencycalculated for different LOH region length cut-offs for the firstcohort. Corresponding log10(p-value) are on the y-axis. The relationshipbetween the cut-off of the size of LOH regions and the significance ofcorrelation of the LOH score with HR deficiency was investigated. Thisfigure shows that LOH length cut-offs may readily range from 11 to 21Mb. The cut-off of 15 Mb, approximately in the middle of the interval,may be used in some preferred embodiments since it was found to be moresensitive to statistical noise present in the data.

FIG. 20 shows comparison of LOH scores in three groups of BRCA1 andBRCA2 deficient samples for the combined data from all three cohorts.Row A: 49 carriers of germline mutations in BRCA1; B: 25 carriers ofsomatic mutations in BRCA1; C: 82 samples with either methylation or lowexpression of BRCA1; D: 27 carriers of germline mutations in BRCA2; E: 9carriers of somatic mutations in BRCA2.

FIG. 21 shows a comparison of LOH scores of BRCA1, BRCA2, and RAD51Cdeficient samples. BRCA1 deficient samples, BRCA2 deficient samples, andRAD51C deficient samples are as indicated. The combined area undercircles for each row is the same. The area under each individual circleis proportional to the number of samples with the corresponding numberof LOH regions.

FIG. 22 shows a comparison of LOH (“HRD”) scores in patients whoresponded versus patients who did not respond to treatment comprisingplatinum therapy. The area under each individual circle is proportionalto the number of samples with the corresponding number of LOH regions.

FIG. 23 shows a comparison of LOH (“HRD”) scores in BRCA1 or BRCA2deficient samples. The area under each individual circle is proportionalto the number of samples with the corresponding number of LOH regions.One outlier sample with significant contamination is highlighted.

FIG. 24 shows the fraction of non-responders in each group of patientswith a given LOH (“HRD”) score.

DETAILED DESCRIPTION

This document provides methods and materials involved in assessingsamples (e.g., cancer cells) for the presence of an LOH signature. Forexample, this document provides methods and materials for determiningwhether or not a cell (e.g., a human cancer cell) contains an LOHsignature (e.g., a HDR-deficiency LOH signature).

In general, a comparison of sequences present at the same locus on eachchromosome (each autosomal chromosome for males) can reveal whether thatparticular locus is homozygous or heterozygous within the genome of acell. Polymorphic loci within the human genome are generallyheterozygous within an individual since that individual typicallyreceives one copy from the biological father and one copy from thebiological mother. In some cases, a polymorphic locus or a string ofpolymorphic loci within an individual are homozygous as a result ofinheriting identical copies from both biological parents.

Loss of heterozygosity (LOH) may result from several mechanisms. Forexample, in some cases, a region of one chromosome can be deleted in asomatic cell. The region that remains present on the other chromosome(the other non-sex chromosome for males) is an LOH region as there isonly one copy (instead of two copies) of that region present within thegenome of the affected cells. This LOH region can be any length (e.g.,from a length less than about 1.5 Mb up to a length equal to the entirelength of the chromosome). This type of LOH event results in a copynumber reduction. In other cases, a region of one chromosome (onenon-sex chromosome for males) in a somatic cell can be replaced with acopy of that region from the other chromosome, thereby eliminating anyheterozygosity that may have been present within the replaced region. Insuch cases, the region that remains present on each chromosome is an LOHregion and can be referred to as a copy neutral LOH region. Copy neutralLOH regions can be any length (e.g., from a length less than about 1.5Mb up to a length equal to the entire length of the chromosome).

As described herein, a cellular sample (e.g., cancer cell sample) can beidentified as having a “positive LOH signature status” (or alternativelycalled “HDR-deficiency LOH signature”) if the genome of the cells beingassessed contains five or more (e.g., six or more, seven or more, eightor more, nine or more, ten or more, eleven or more, 12 or more, 13 ormore, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 ormore, or 20 or more) LOH regions that are (a) longer than about 1.5megabases (e.g., longer than about 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, or 100megabases (Mb), preferably longer than about 14 or 15 or 16, morepreferably longer than about 15 megabases) and (b) less than the lengthof the entire chromosome that contains that LOH region. In some cases, acancer cell sample can be identified as having a positive LOH signaturestatus if the genome of the cells being assessed contains nine or moreLOH regions that are (a) longer than about 15 Mb and (b) less than thelength of the entire chromosome that contains that LOH region. Unlessotherwise defined, the term “Indicator LOH Region” refers to an LOHregion that is in a pair of human chromosomes other than the human X/Ysex chromosome pair, and that is characterized by loss of heterozygositywith a length of about 1.5 or more megabases but shorter than the lengthof the whole chromosome containing the LOH region. The length of thewhole chromosome containing an LOH region may be determined by examiningthe length of the shorter chromosome of the corresponding chromosomepair in a germline cell or a non-tumor somatic cell. In someembodiments, an Indicator LOH Region is any LOH region about 2, 2.5, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, 75, or 100 megabases (Mb) or more (preferably longerthan about 14 or 15 megabases) and less than the length of the wholechromosome that contains that LOH region.

Cells (e.g., cancer cells) identified as having a positive LOH signature(also termed herein “HDR-deficiency LOH signature”) can be classified ashaving an increased likelihood of having an HDR deficiency and/or ashaving an increased likelihood of having a deficient status in one ormore genes in the HDR pathway. For example, cancer cells identified ashaving a positive LOH signature status can be classified as having anincreased likelihood of having an HDR deficient status. In some cases,cancer cells identified as having a positive LOH signature status can beclassified as having an increased likelihood of having a deficientstatus for one or more genes in the HDR pathway. As used herein,deficient status for a gene means the sequence, structure, expressionand/or activity of the gene or its product is/are deficient as comparedto normal. Examples include, but are not limited to, low or no mRNA orprotein expression, deleterious mutations, hypermethylation, attenuatedactivity (e.g., enzymatic activity, ability to bind to anotherbiomolecule), etc. As used herein, deficient status for a pathway (e.g.,HDR pathway) means at least one gene in that pathway (e.g., BRCA1) isdeficient. Examples of highly deleterious mutations include frameshiftmutations, stop codon mutations, and mutations that lead to altered RNAsplicing. Deficient status in a gene in the HDR pathway may result indeficiency or reduced activity in homology directed repair in the cancercells. Examples of genes in the HDR pathway include, without limitation,the genes listed in Table 1.

TABLE 1 Selected HDR Pathway Genes Gene Name Entrez Gene Symbol (ifassigned) Entrez Gene Id Gene Name Entrez Gene Symbol (if assigned)Entrez Gene Id BLM BLM 641 RAD50 RAD50 10111 BRCA1 BRCA1 672 RAD51 RAD515888 BRCA2 BRCA2 675 RAD51AP1 RAD51AP1 10635 CtIP RBBP8 5932 RAD51BRAD51L1 5890 DNA polymerase delta POLD1 5424 RAD51C RAD51C 5889 POLD25424 RAD51D RAD51L3 5892 POLD3 10714 RAD54 ATRX 546 POLD4 57804 RAD54BRAD54B 25788 DNA polymerase eta POLH 5429 RMI1 RMI1 80010 DNA2 DNA2 1763RMI2 C16orf75 116028 EME1 EME1 146956 RPA RPA1 6117 ERCC1 ERCC1 2067RTEL1 RTEL1 51750 EXO1 EXO1 9156 SLX1 FANCM FANCM 57697 SLX2 GEN1 GEN1348654 SLX4 SLX4 84464 MRE11 MRE11A 4361 TOP2A TOP2A 7153 MUS81 MUS8180198 XPF ERCC4 2072 NBS1 NBN 4683 XRCC2 XRCC2 7516 PALB2 PALB2 79728XRCC3 XRCC3 7517 PCNA PCNA 5111

Examples of genetic mutations that can be present within a gene of theHDR pathway include, without limitation, those listed in Table 2.

TABLE 2 Possible genetic mutations within selected genes of the HDRpathway Gene Mutation Entrez Gene ID BRCA1 C24F 672 BRCA1 E29X 672 BRCA2R3052W 675 BRCA2 2881delG 675 RAD51C G125V 5889 RAD51C L138F 5889 RAD51CY75XfsX0 5889

In some cases, a cellular sample (e.g., cancer cell sample) can beidentified as having an increased number of LOH regions (e.g., at least7, 8, 9, 10, or more LOH regions) that cover the whole chromosome. Cells(e.g., cancer cells) identified as having an increased number of LOHregions that cover the whole chromosome can be classified as having anincreased likelihood of having HDR proficiency, that is, intact HDRpathway. For example, cancer cells identified as having an increasednumber of LOH regions that cover the whole chromosome can be classifiedas being more likely to have intact BRCA1 and BRCA2 genes.

As described herein, identifying LOH loci (as well as the size andnumber of LOH regions) can include, first, determining the genotype of asample at various genomic loci (e.g., SNP loci, individual bases inlarge sequencing) and, second, determining whether homozygous loci aredue to LOH events. Any appropriate technique can be used to determinegenotypes at loci of interest within the genome of a cell. For example,single nucleotide polymorphisms (SNP) arrays (e.g., human genome-wideSNP arrays), targeted sequencing of loci of interest (e.g., sequencingSNP loci and their surrounding sequences), and even untargetedsequencing (e.g., whole exome, transcriptome, or genome sequencing) canbe used to identify loci as being homozygous or heterozygous. In somecases, an analysis of the homozygous or heterozygous nature of loci overa length of a chromosome can be performed to determine the length ofregions of homozygosity or heterozygosity. For example, a stretch of SNPlocations that are spaced apart (e.g., spaced about 25 kb to about 100kb apart) along a chromosome can be evaluated using SNP array results todetermine not only the presence of a region of homozygosity along achromosome but also the length of that region. Results from a SNP arraycan be used to generate a graph that plots allele dosages along achromosome. Allele dosage d_(i) for SNP i can be calculated fromadjusted signal intensities of two alleles (A_(i) and B_(i)): d_(i) =A_(i)/(A_(i) + B_(i)). An example of such a graph is presented in FIG. 1. Numerous variations on nucleic acid arrays useful in the invention areknown in the art. These include the arrays used in the various examplesbelow (e.g., Affymetrix 500K GeneChip array in Example 3; AffymetrixOncoScan™ FFPE Express 2.0 Services (Formerly MIP CN Services) inExample 4).

Once a sample’s genotype has been determined for a plurality of loci(e.g., SNPs), common techniques can be used to identify loci and regionsof LOH. One way to determine whether homozygosity is due to LOH is tocompare the somatic genotype to the germline. For example, the genotypefor a plurality of loci (e.g., SNPs) can be determined in both agermline (e.g., blood) sample and a somatic (e.g., tumor) sample. Thegenotypes for each sample can be compared (typically computationally) todetermine where the genome of the germline cell was heterozygous and thegenome of the somatic cell is homozygous. Such loci are LOH loci andregions of such loci are LOH regions.

Computational techniques can also be used to determine whetherhomozygosity is due to LOH. Such techniques are particularly useful whena germline sample is not available for analysis and comparison. Forexample, algorithms such as those described elsewhere can be used todetect LOH regions using information from SNP arrays (Nannya et al.,Cancer Res. (2005) 65:6071-6079 (2005)). Typically these algorithms donot explicitly take into account contamination of tumor samples withbenign tissue. Cf. International Application No. PCT/US2011/026098 toAbkevich et al.; Goransson et al., PLoS One (2009) 4(6):e6057. Thiscontamination is often high enough to make the detection of LOH regionschallenging. Improved analytical methods according to the presentinvention for identifying LOH, even in spite of contamination, includethose embodied in computer software products as described below.

The following is one example. If the observed ratio of the signals oftwo alleles, A and B, is two to one, there are two possibilities. Thefirst possibility is that cancer cells have LOH with deletion of alleleB in a sample with 50% contamination with normal cells. The secondpossibility is that there is no LOH but allele A is duplicated in asample with no contamination with normal cells. An algorithm can beimplemented as a computer program as described herein to reconstruct LOHregions based on genotype (e.g., SNP genotype) data. One point of thealgorithm is to first reconstruct allele specific copy numbers (ASCN) ateach locus (e.g., SNP). ASCNs are the numbers of copies of both paternaland maternal alleles. An LOH region is then determined as a stretch ofSNPs with one of the ASCNs (paternal or maternal) being zero. Thealgorithm can be based on maximizing a likelihood function and can beconceptually akin to a previously described algorithm designed toreconstruct total copy number (rather than ASCN) at each locus (e.g.,SNP). See International Application No. PCT/US2011/026098 to Abkevich etal. The likelihood function can be maximized over ASCN of all loci,level of contamination with benign tissue, total copy number averagedover the whole genome, and sample specific noise level. The input datafor the algorithm can include or consist of (1) sample-specificnormalized signal intensities for both allele of each locus and (2)assay-specific (specific for different SNP arrays and for sequence basedapproach) set of parameters defined based on analysis of large number ofsamples with known ASCN profiles.

In some cases, nucleic acid sequencing techniques can be used toidentify loci as being homozygous or heterozygous. For example, genomicDNA from a cell sample (e.g., a cancer cell sample) can be extracted andfragmented. Any appropriate method can be used to extract and fragmentgenomic nucleic acid including, without limitation, commercial kits suchas QIAamp™ DNA Mini Kit (Qiagen™), MagNA™ Pure DNA Isolation Kit (RocheApplied Science™) and GenElute™ Mammalian Genomic DNA Miniprep Kit(Sigma-Aldrich™). Once extracted and fragmented, either targeted oruntargeted sequencing can be done to determine the sample’s genotypes atloci. For example, whole genome, whole transcriptome, or whole exomesequencing can be done to determine genotypes at millions or evenbillions of base pairs (i.e., base pairs can be “loci” to be evaluated).

In some cases, targeted sequencing of known polymorphic loci (e.g., SNPsand surrounding sequences) can be done as an alternative to microarrayanalysis. For example, the genomic DNA can be enriched for thosefragments containing a locus (e.g., SNP location) to be analyzed usingkits designed for this purpose (e.g., Agilent SureSelect™, IlluminaTruSeq Capture™, and Nimblegen SeqCap EZ Choice™). For example, genomicDNA containing the loci to be analyzed can be hybridized to biotinylatedcapture RNA fragments to form biotinylated RNA/genomic DNA complexes.Alternatively, DNA capture probes may be utilized resulting in theformation of biotinylated DNA/genomic DNA hybrids. Streptavidin coatedmagnetic beads and a magnetic force can be used to separate thebiotinylated RNA/genomic DNA complexes from those genomic DNA fragmentsnot present within a biotinylated RNA/genomic DNA complex. The obtainedbiotinylated RNA/genomic DNA complexes can be treated to remove thecaptured RNA from the magnetic beads, thereby leaving intact genomic DNAfragments containing a locus to be analyzed. These intact genomic DNAfragments containing the loci to be analyzed can be amplified using, forexample, PCR techniques. The amplified genomic DNA fragments can besequenced using a high-throughput sequencing technology or anext-generation sequencing technology such as Illumina HiSeq™, IlluminaMiSeq™, Life Technologies SoLID™ or Ion Torrent™, or Roche 454™.

The sequencing results from the genomic DNA fragments can be used toidentify loci as being homozygous or heterozygous, analogous to themicroarray analysis described herein. In some cases, an analysis of thehomozygous or heterozygous nature of loci over a length of a chromosomecan be performed to determine the length of regions of homozygosity orheterozygosity. For example, a stretch of SNP locations that are spacedapart (e.g., spaced about 25 kb to about 100 kb apart) along achromosome can be evaluated by sequencing, and the sequencing resultsused to determine not only the presence of a region of homozygosityalong a chromosome but also the length of that LOH region. Obtainedsequencing results can be used to generate a graph that plots alleledosages along a chromosome. Allele dosage d_(i) for SNP i can becalculated from adjusted number of captured probes for two alleles(A_(i) and B_(i)): d_(i) = A_(i)/(A_(i) + Bi). An example of such agraph is presented in FIG. 2 . Determining whether homozygosity is dueto LOH (as opposed to homozygosity in the germline) can be performed asdescribed herein.

In some cases, a selection process can be used to select loci (e.g., SNPloci) to be evaluated using an assay configured to identify loci asbeing homozygous or heterozygous (e.g., SNP array-based assays andsequencing-based assays). For example, any human SNP location can beselected for inclusion in a SNP array-based assay or a sequencing-basedassay configured to identify loci as being homozygous or heterozygouswithin the genome of cells. In some cases, 0.5, 1.0, 1.5, 2.0, 2.5million or more SNP locations present within the human genome can beevaluated to identify those SNPs that (a) are not present on the Ychromosome, (b) are not mitochondrial SNPs, (c) have a minor allelefrequency of at least about five percent in Caucasians, (d) have a minorallele frequency of at least about one percent in three races other thanCaucasians (e.g., Chinese, Japanese, and Yoruba), and/or (e) do not havea significant deviation from Hardy Weinberg equilibrium in any of thefour races. In some cases, more than 100,000, 150,000, or 200,000 humanSNPs can be selected that meet criteria (a) through (e). Of the humanSNPs meeting criteria (a) through (e), a group of SNPs (e.g., top110,000 SNPs) can be selected such that the SNPs have a high degree ofallele frequency in Caucasians, cover the human genome in a somewhatevenly spaced manner (e.g., at least one SNP every about 25 kb to about500 kb), and are not in linkage disequilibrium with another selected SNPfor in any of the four races. In some cases, about 40, 50, 60, 70, 80,90, 100, 110, 120, 130 thousand or more SNPs can be selected as meetingeach of these criteria and included in an assay configured to identifyLOH regions across a human genome. For example, between about 70,000 andabout 90,000 (e.g., about 80,000) SNPs can be selected for analysis witha SNP array-based assay, and between about 45,000 and about 55,000(e.g., about 54,000) SNPs can be selected for analysis with asequencing-based assay.

As described herein, a cell sample can be assessed to determine if thegenome of cells of the sample contains an LOH signature, lacks an LOHsignature, has an increased number of LOH regions that cover the wholechromosome, or lacks an increased number of LOH regions that cover thewhole chromosome. Any appropriate type of sample can be assessed. Forexample, a sample containing cancer cells can be assessed to determineif the genome of the cancer cells contains an LOH signature, lacks anLOH signature, has an increased number of LOH regions that cover thewhole chromosome, or lacks an increased number of LOH regions that coverthe whole chromosome. Examples of samples containing cancer cells thatcan be assessed as described herein include, without limitation, tumorbiopsy samples (e.g., breast tumor biopsy samples), formalin-fixed,paraffin-embedded tissue samples containing cancer cells, core needlebiopsies, fine needle aspirates, and samples containing cancer cellsshed from a tumor (e.g., blood, urine or other bodily fluids). Forformalin-fixed, paraffin-embedded tissue samples, the sample can beprepared by DNA extraction using a genomic DNA extraction kit optimizedfor FFPE tissue, including but not limited to those described above(e.g., QuickExtract™ FFPE DNA Extraction Kit (Epicentre™), and QIAamp™DNA FFPE Tissue Kit (Qiagen™)).

In some cases, laser dissection techniques can be performed on a tissuesample to minimize the number of non-cancer cells within a cancer cellsample to be assessed. In some cases, antibody based purificationmethods can be used to enrich for cancer cells and/or deplete non-cancercells. Examples of antibodies that could be used for cancer cellenrichment include, without limitation, anti-EpCAM, anti-TROP-2,anti-c-Met, anti-Folate binding protein, anti-N-Cadherin, anti-CD318,anti-antimesencymal stem cell antigen, anti-Her2, anti-MUC1, anti-EGFR,anti-cytokeratins (e.g., cytokeratin 7, cytokeratin 20, etc.),anti-Caveolin-1, anti-PSA, anti-CA125, and anti-surfactant proteinantibodies.

Any type of cancer cell can be assessed using the methods and materialsdescribed herein. For example, breast cancer cells, ovarian cancercells, liver cancer cells, esophageal cancer cells, lung cancer cells,head and neck cancer cells, prostate cancer cells, colon, rectal, orcolorectal cancer cells, and pancreatic cancer cells can be assessed todetermine if the genome of the cancer cells contains an LOH signature,lacks an LOH signature, has an increased number of LOH regions thatcover the whole chromosome, or lacks an increased number of LOH regionsthat cover the whole chromosome. In some embodiments, the cancer cellsare primary or metastatic cancer cells of ovarian cancer, breast cancer,lung cancer or esophageal cancer.

When assessing the genome of cancer cells for the presence or absence ofan LOH signature, one or more (e.g., one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, or 23) pairs of chromosomes can be assessed. In some cases, thegenome of cancer cells is assessed for the presence or absence of an LOHsignature using one or more (e.g., one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23) pairs of chromosomes.

In some cases, it can be helpful to exclude certain chromosomes fromthis analysis. For example, in the case of females, a pair to beassessed can include the pair of X sex chromosomes; whereas, in the caseof males, a pair of any autosomal chromosomes (i.e., any pair other thanthe pair of X and Y sex chromosomes) can be assessed. As anotherexample, in some cases the chromosome number 17 pair may be excludedfrom the analysis. It has been determined that certain chromosomes carryunusually high levels of LOH in certain cancers and, thus, it can behelpful to exclude such chromosomes when analyzing samples as describedherein from patients having these cancers. In some cases, the sample isfrom a patient having ovarian cancer, and the chromosome to be excludedis chromosome 17.

When assessing the genome of cancer cells for the presence or absence ofan increased number of LOH regions that cover the whole chromosome, 10or more (e.g., 13, 16, 19 or 23) pairs of chromosomes can be assessed.In the case of females, a pair to be assessed can include the pair of Xsex chromosomes; whereas, in the case of males, a pair of any autosomalchromosomes (i.e., any pair other than the pair of X and Y sexchromosomes) can be assessed. In some cases, the chromosome number 17pair may be excluded from the analysis. In some cases, the sample isfrom a patient having ovarian cancer, and the chromosome to be excludedis chromosome 17. In some cases, the genome of cancer cells is assessedfor the presence or absence of an increased number of LOH regions thatcover the whole chromosome using 10 or more (e.g., 13, 16, 19, or 23)pairs of chromosomes.

Thus, a predefined number of chromosomes may be analyzed to determinethe total number of Indicator LOH Regions, preferably the total numberof LOH regions of a length of greater than 9 megabases, 10 megabases, 12megabases, 14 megabases, more preferably greater than 15 megabases.Alternatively or in addition, the sizes of all identified Indicator LOHRegions may be summed up to obtain a total length of Indicator LOHRegions.

For classification of positive LOH signature status, the referencenumber discussed above for the total number of Indicator LOH Regions maybe 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20 or greater,preferably 5, preferably 8, more preferably 9 or 10, most preferably 10.The reference number for the total (e.g., combined) length of IndicatorLOH Regions may be about 75, 90, 105, 120, 130, 135, 150, 175, 200, 225,250, 275, 300, 325 350, 375, 400, 425, 450, 475, 500 megabases orgreater, preferably about 75 megabases or greater, preferably about 90or 105 megabases or greater, more preferably about 120 or 130 megabasesor greater, and more preferably about 135 megabases or greater, and mostpreferably about 150 megabases or greater.

In some specific embodiments, the total number of LOH regions of alength of greater than about 14 or 15 megabases is determined andcompared to a reference number of about 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 18, 19, or 20. Alternatively or in addition, the totallength of LOH regions of a length of greater than about 14 or 15megabases is determined and compared to a reference number of about 75,90, 105, 120, 130, 135, 150, 175, 200, 225, 250, 275, 300, 325 350, 375,400, 425, 450, 475, or 500 megabases.

In some embodiments, the number of LOH regions (or the combined length,or a test value or score derived from either) in a patient sample isconsidered “greater” than a reference if it is at least 2-, 3-, 4-, 5-,6-, 7-, 8-, 9-, or 10-fold greater than the reference while in someembodiments, it is considered “greater” if it is at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 standard deviations greater than the reference.Conversely, in some embodiments the number of LOH regions (or thecombined length, or a test value or score derived from either) in apatient sample is considered “not greater” than a reference if it is notmore than 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold greater than thereference while in some embodiments, it is considered “not greater” ifit is not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 standard deviationsgreater than the reference.

In some embodiments the reference number (or length, value or score) isderived from a relevant reference population. Such reference populationsmay include patients (a) with the same cancer as the patient beingtested, (b) with the same cancer sub-type, (c) with cancer havingsimilar genetic or other clinical or molecular features, (d) whoresponded to a particular treatment, (e) who did not respond to aparticular treatment, (f) who are apparently healthy (e.g., do not haveany cancer or at least do not have the tested patient’s cancer), etc.The reference number (or length, value or score) may be (a)representative of the number (or length, value or score) found in thereference population as a whole, (b) an average (mean, median, etc.) ofthe number (or length, value or score) found in the reference populationas a whole or a particular subpopulation, (c) representative of thenumber (or length, value or score) (e.g., an average such as mean ormedian) found in terciles, quartiles, quintiles, etc. of the referencepopulation as ranked by (i) their respective number (or length, value orscore) or (ii) the clinical feature they were found to have (e.g.,strength of response, prognosis (including time to cancer-specificdeath), etc.).

As described herein, patients having cancer cells identified as having apositive LOH signature status can be classified, based at least in parton a positive LOH signature status, as being likely to respond to aparticular cancer treatment regimen. For example, patients having cancercells with a genome containing an LOH signature can be classified, basedat least in part on a positive LOH signature status, as being likely torespond to a cancer treatment regimen that includes the use of a DNAdamaging agent, a synthetic lethality agent (e.g., a PARP inhibitor),radiation, or a combination thereof. Preferably the patients aretreatment naïve patients. Examples of DNA damaging agents include,without limitation, platinum-based chemotherapy drugs (e.g., cisplatin,carboplatin, oxaliplatin, and picoplatin), anthracyclines (e.g.,epirubicin and doxorubicin), topoisomerase I inhibitors (e.g.,campothecin, topotecan, and irinotecan), DNA crosslinkers such asmitomycin C, and triazene compounds (e.g., dacarbazine andtemozolomide). Synthetic lethality therapeutic approaches typicallyinvolve administering an agent that inhibits at least one criticalcomponent of a biological pathway that is especially important to aparticular tumor cell’s survival. For example, when a tumor cell has adeficient homologous repair pathway (e.g., as determined according tothe present invention), inhibitors of poly ADP ribose polymerase (orplatinum drugs, double strand break repair inhibitors, etc.) can beespecially potent against such tumors because two pathways critical tosurvival become obstructed (one biologically, e.g., by BRCA1 mutation,and the other synthetically, e.g., by administration of a pathway drug).Synthetic lethality approaches to cancer therapy are described in, e.g.,O’Brien et al., Converting cancer mutations into therapeuticopportunities, EMBO MOL. MED. (2009) 1:297-299. Examples of syntheticlethality agents include, without limitation, PARP inhibitors or doublestrand break repair inhibitors in homologous repair-deficient tumorcells, PARP inhibitors in PTEN-deficient tumor cells, methotrexate inMSH2-deficient tumor cells, etc. Examples of PARP inhibitors include,without limitation, olaparib, iniparib, and veliparib. Examples ofdouble strand break repair inhibitors include, without limitation,KU55933 (ATM inhibitor) and NU7441 (DNA-PKcs inhibitor). Examples ofinformation that can be used in addition to a positive LOH signaturestatus to base a classification of being likely to respond to aparticular cancer treatment regimen include, without limitation,previous treatment results, germline or somatic DNA mutations, gene orprotein expression profiling (e.g., ER/PR/HER2 status, PSA levels),tumor histology (e.g., adenocarcinoma, squamous cell carcinoma,papillary serous carcinoma, mucinous carcinoma, invasive ductalcarcinoma, ductal carcinoma in situ (non-invasive), etc.), diseasestage, tumor or cancer grade (e.g., well, moderately, or poorlydifferentiated (e.g., Gleason, modified Bloom Richardson), etc.), numberof previous courses of treatment, etc.

Once classified as being likely to respond to a particular cancertreatment regimen (e.g., a cancer treatment regimen that includes theuse of a DNA damaging agent, a PARP inhibitor, radiation, or acombination thereof), the cancer patient can be treated with such acancer treatment regimen. In some embodiments, the patients aretreatment naïve patients. Any appropriate method for treating the cancerat issue can be used to treat a cancer patient identified as havingcancer cells having a positive LOH signature status. For example,platinum-based chemotherapy drugs or a combination of platinum-basedchemotherapy drugs can be used to treat cancer as described elsewhere(see, e.g., U.S. Pat. No. 3,892,790, 3,904,663, 7,759,510, 7,759,488 and7,754,684. In some cases, anthracyclines or a combination ofanthracyclines can be used to treat cancer as described elsewhere (see,e.g., U.S. Pat. No. 3,590,028, 4,138,480, 4,950,738, 6,087,340,7,868,040, and 7,485,707. In some cases, topoisomerase I inhibitors or acombination of topoisomerase I inhibitors can be used to treat cancer asdescribed elsewhere (see, e.g., U.S. Pat. No. 5,633,016 and 6,403,563.In some cases, PARP inhibitors or a combination of PARP inhibitors canbe used to treat cancer as described elsewhere (see, e.g., U.S. Pat. No.5,177,075, 7,915,280, and 7,351,701. In some cases, radiation can beused to treat cancer as described elsewhere (see, e.g., U.S. Pat. No.5,295,944). In some cases, a combination comprising different agents(e.g., a combination comprising any of platinum-based chemotherapydrugs, anthracyclines, topoisomerase I inhibitors, and/or PARPinhibitors) with or without radiation treatments can be used to treatcancer. In some cases, a combination treatment may comprise any of theabove agents or treatments (e.g., a DNA damaging agent, a PARPinhibitor, radiation, or a combination thereof) together with anotheragent or treatment—e.g., a taxane agent (e.g., doxetaxel, paclitaxel,abraxane), a growth factor or growth factor receptor inhibitor (e.g.,erlotinib, gefitinib, lapatinib, sunitinib, bevacizumab, cetuximab,trastuzumab, panitumumab), and/or an antimetabolite (e.g.,5-flourouracil, methotrexate).

In some cases, patients identified as having cancer cells with a genomelacking an LOH signature can be classified, based at least in part on anegative LOH signature status, as being less likely to respond to atreatment regimen that includes a DNA damaging agent, a PARP inhibitor,radiation, or a combination thereof. In turn, such a patient can beclassified as likely to respond to a cancer treatment regimen thatincludes the use of one or more cancer treatment agents not associatedwith HDR, such as a taxane agent (e.g., doxetaxel, paclitaxel,abraxane), a growth factor or growth factor receptor inhibitor (e.g.,erlotinib, gefitinib, lapatinib, sunitinib, bevacizumab, cetuximab,trastuzumab, panitumumab), and/or an antimetabolite agent (e.g.,5-flourouracil, methotrexate). In some embodiments, the patients aretreatment naïve patients. Once classified as being likely to respond toa particular cancer treatment regimen (e.g., a cancer treatment regimenthat includes the use of a cancer treatment agent not associated withHDR), the cancer patient can be treated with such a cancer treatmentregimen. Any appropriate method for the cancer being treated can be usedto treat a cancer patient identified as having cancer cells having anegative LOH signature status. Examples of information that can be usedin addition to a negative LOH signature status to base a classificationof being likely to respond to a particular cancer treatment regimeninclude, without limitation, previous treatment results, germline orsomatic DNA mutations, gene or protein expression profiling (e.g.,ER/PR/HER2 status, PSA levels), tumor histology (e.g., adenocarcinoma,squamous cell carcinoma, papillary serous carcinoma, mucinous carcinoma,invasive ductal carcinoma, ductal carcinoma in situ (non-invasive),etc.), disease stage, tumor or cancer grade (e.g., well, moderately, orpoorly differentiated (e.g., Gleason, modified Bloom Richardson), etc.),number of previous courses of treatment, etc.

Once treated for a particular period of time (e.g., between one to sixmonths), the patient can be assessed to determine whether or not thetreatment regimen has an effect. If a beneficial effect is detected, thepatient can continue with the same or a similar cancer treatmentregimen. If a minimal or no beneficial effect is detected, thenadjustments to the cancer treatment regimen can be made. For example,the dose, frequency of administration, or duration of treatment can beincreased. In some cases, additional anti-cancer agents can be added tothe treatment regimen or a particular anti-cancer agent can be replacedwith one or more different anti-cancer agents. The patient being treatedcan continue to be monitored as appropriate, and changes can be made tothe cancer treatment regimen as appropriate.

In addition to predicting likely treatment response or selectingdesirable treatment regimens, an LOH signature can be used to determinea patient’s prognosis. As shown in Example 3 below (particularly FIG. 18b ), patients whose tumors have an LOH signature show significantlybetter survival than patients whose tumors do not have such an LOHsignature. Thus, in one aspect, this document features a method fordetermining a patient’s prognosis based at least in part of detectingthe presence or absence of an LOH signature in a sample from thepatient. The method comprises, or consists essentially of, (a)determining whether the patient comprises cancer cells having an LOHsignature as described herein (e.g., wherein the presence of more than areference number of LOH regions in at least one pair of humanchromosomes of a cancer cell of the cancer patient that are longer thana first length but shorter than the length of the whole chromosomecontaining the LOH region indicates that the cancer cells have the LOHsignature, wherein the at least one pair of human chromosomes is not ahuman X/Y sex chromosome pair, wherein the first length is about 1.5 ormore megabases), and (b)(1) determining, based at least in part on thepresence of the LOH signature, that the patient has a relatively goodprognosis, or (b)(2) determining, based at least in part on the absenceof the LOH signature, that the patient has a relatively poor prognosis.Prognosis may include the patient’s likelihood of survival (e.g.,progression-free survival, overall survival), wherein a relatively goodprognosis would include an increased likelihood of survival as comparedto some reference population (e.g., average patient with this patient’scancer type/subtype, average patient not having an LOH signature, etc.).Conversely, a relatively poor prognosis in terms of survival wouldinclude a decreased likelihood of survival as compared to some referencepopulation (e.g., average patient with this patient’s cancertype/subtype, average patient having an LOH signature, etc.).

As described herein, this document provides methods for assessingpatients for cells (e.g., cancer cells) having a genome containing anLOH signature. In some embodiments, the patients are treatment naïvepatients. For example, one or more clinicians or medical professionalscan determine if a patient contains cancer cells having a genomecontaining an LOH signature. In some cases, one or more clinicians ormedical professionals can determine if a patient contains cancer cellshaving a genome containing an LOH signature by obtaining a cancer cellsample from the patient and assessing the genome of cancer cells of thecancer cell sample to determine the presence or absence of an LOHsignature as described herein.

In some cases, one or more clinicians or medical professionals canobtain a cancer cell sample from a patient and provide that sample to atesting laboratory having the ability to assess the genome of cancercells of the cancer cell sample to provide an indication about thepresence or absence of an LOH signature as described herein. In someembodiments, the patients are treatment naïve patients. In such cases,the one or more clinicians or medical professionals can determine if apatient contains cancer cells having a genome containing an LOHsignature by receiving information about the presence or absence of anLOH signature directly or indirectly from the testing laboratory. Forexample, a testing laboratory, after assessing the genome of cancercells for presence or absence of an LOH signature as described herein,can provide a clinician or medical professional with, or access to, awritten, electronic, or oral report or medical record that provides anindication about the presence or absence of an LOH signature for aparticular patient being assessed. Such a written, electronic, or oralreport or medical record can allow the one or more clinicians or medicalprofessionals to determine if a particular patient being assessedcontains cancer cells having a genome containing an LOH signature.

Once a clinician or medical professional or group of clinicians ormedical professionals determines that a particular patient beingassessed contains cancer cells having a genome containing an LOHsignature, the clinician or medical professional (or group) can classifythat patient as having cancer cells whose genome contains the presenceof an LOH signature. In some embodiments, the patients are treatmentnaïve patients. In some cases, a clinician or medical professional orgroup of clinicians or medical professionals can diagnose a patientdetermined to have cancer cells whose genome contains the presence of anLOH signature as having cancer cells likely to be deficient in HDR. Sucha diagnosis can be based solely on a determination that a particularpatient being assessed contains cancer cells having a genome containingan LOH signature or can be based at least in part on a determinationthat a particular patient being assessed contains cancer cells having agenome containing an LOH signature. For example, a patient determined tohave cancer cells whose genome contains the presence of an LOH signaturecan be diagnosed as likely to be deficient in HDR based on thecombination of a positive LOH signature status and deficient status inone or more tumor suppressor genes (e.g., BRCA1/2, RAD51C), a familyhistory of cancer, or the presence of behavioral risk factors (e.g.,smoking).

In some cases, a clinician or medical professional or group ofclinicians or medical professionals can diagnose a patient determined tohave cancer cells whose genome contains the presence of an LOH signatureas having cancer cells likely to contain genetic mutations in one ormore genes in the HDR pathway. In some embodiments, the patients aretreatment naïve patients. Such a diagnosis can be based solely on adetermination that a particular patient being assessed contains cancercells having a genome containing an LOH signature or can be based atleast in part on a determination that a particular patient beingassessed contains cancer cells having a genome containing an LOHsignature. For example, a patient determined to have cancer cells whosegenome contains the presence of an LOH signature can be diagnosed ashaving cancer cells likely to contain genetic mutations in one or moregenes in the HDR pathway based on the combination of a positive LOHpositive status and a family history of cancer, or the presence ofbehavioral risk factors (e.g., smoking).

In some cases, a clinician or medical professional or group ofclinicians or medical professionals can diagnose a patient determined tohave cancer cells whose genome contains the presence of an LOH signatureas having cancer cells likely to respond to a particular cancertreatment regimen. In some embodiments, the patients are treatment naïvepatients. Such a diagnosis can be based solely on a determination that aparticular patient being assessed contains cancer cells having a genomecontaining an LOH signature or can be based at least in part on adetermination that a particular patient being assessed contains cancercells having a genome containing an LOH signature. For example, apatient determined to have cancer cells whose genome contains thepresence of an LOH signature can be diagnosed as being likely to respondto a particular cancer treatment regimen based on the combination of apositive LOH signature status and deficient status in one or more tumorsuppressor genes (e.g., BRCA1/2, RAD51), a family history of cancer, orthe presence of behavioral risk factors (e.g., smoking). As describedherein, a patient determined to have cancer cells whose genome containsthe presence of an LOH signature can be diagnosed as likely to respondto a cancer treatment regimen that includes the use of a platinum-basedchemotherapy drug such as cisplatin, carboplatin, oxaliplatin, orpicoplatin, an anthracycline such as epirubicin or doxorubicin, atopoisomerase I inhibitor such as campothecin, topotecan, or irinotecan,a PARP inhibitor, radiation, a combination thereof, or a combination ofany of the preceding with another anti-cancer agent. In someembodiments, the patients are treatment naïve patients.

Once a clinician or medical professional or group of clinicians ormedical professionals determines that a particular patient beingassessed contains cancer cells having a genome lacking an LOH signature,the clinician or medical professional (or group) can classify thatpatient as having cancer cells whose genome contains an absence of anLOH signature. In some embodiments, the patients are treatment naïvepatients. In some cases, a clinician or medical professional or group ofclinicians or medical professionals can diagnose a patient determined tohave cancer cells containing a genome that lacks the presence of an LOHsignature as having cancer cells likely to have functional HDR. In somecases, a clinician or medical professional or group of clinicians ormedical professionals can diagnose a patient determined to have cancercells containing a genome that lacks the presence of an LOH signature ashaving cancer cells that do not likely contain genetic mutations in oneor more genes in the HDR pathway. In some cases, a clinician or medicalprofessional or group of clinicians or medical professionals candiagnose a patient determined to have cancer cells containing a genomethat lacks the presence of an LOH signature or contains an increasednumber of LOH regions that cover the whole chromosome as having cancercells that are less likely to respond to a platinum-based chemotherapydrug such as cisplatin, carboplatin, oxalaplatin, or picoplatin, ananthracycline such as epirubincin or doxorubicin, a topoisomerase Iinhibitor such as campothecin, topotecan, or irinotecan, a PARPinhibitor, or radiation and/or more likely to respond to a cancertreatment regimen that includes the use of a cancer treatment agent notassociated with HDR such as one or more taxane agents, growth factor orgrowth factor receptor inhibitors, anti-metabolite agents, etc. In someembodiments, the patients are treatment naïve patients.

As described herein, this document also provides methods for performinga diagnostic analysis of a nucleic acid sample (e.g., a genomic nucleicacid sample or amplified genomic nucleic acid sample) of a cancerpatient to determine if cancer cells within the patient have a genomecontaining an LOH signature and/or an increased number of LOH regionsthat cover the whole chromosome. In some embodiments, the patients aretreatment naïve patients. For example, one or more laboratorytechnicians or laboratory professionals can detect the presence orabsence of an LOH signature in the genome of cancer cells of the patientor the presence or absence of an increased number of LOH regions thatcover the whole chromosome in the genome of cancer cells of the patient.In some cases, one or more laboratory technicians or laboratoryprofessionals can detect the presence or absence of an LOH signature orthe presence or absence of an increased number of LOH regions that coverthe whole chromosome in the genome of cancer cells of the patient by (a)receiving a cancer cell sample obtained from the patient, receiving agenomic nucleic acid sample obtained from cancer cells obtained from thepatient, or receiving an enriched and/or amplified genomic nucleic acidsample obtained from cancer cells obtained from the patient and (b)performing an analysis (e.g., a SNP array-based assay or asequencing-based assay) using the received material to detect thepresence or absence of an LOH signature or the presence or absence of anincreased number of LOH regions that cover the whole chromosome asdescribed herein. In some cases, one or more laboratory technicians orlaboratory professionals can receive a sample to be analyzed (e.g., acancer cell sample obtained from the patient, a genomic nucleic acidsample obtained from cancer cells obtained from the patient, or anenriched and/or amplified genomic nucleic acid sample obtained fromcancer cells obtained from the patient) directly or indirectly from aclinician or medical professional. In some embodiments, the patients aretreatment naïve patients.

Once a laboratory technician or laboratory professional or group oflaboratory technicians or laboratory professionals detects the presenceof an LOH signature as described herein, the laboratory technician orlaboratory professional (or group) can identify the patient whose cancercells were detected as having an LOH signature as having cancer cellswith a positive LOH signature status. For example, one or morelaboratory technicians or laboratory professionals can identify apatient having cancer cells that were detected to have an LOH signatureas having cancer cells with a positive LOH signature status byassociating that positive LOH signature status or the result (or resultsor a summary of results) of the performed diagnostic analysis with thecorresponding patient’s name, medical record, symbolic/numericalidentifier, or a combination thereof. In some cases, a laboratorytechnician or laboratory professional or group of laboratory techniciansor laboratory professionals can identify a patient having cancer cellsthat were detected to have an LOH signature as having cancer cellspotentially deficient in HDR by associating the positive LOH signaturestatus, the potentially deficient in HDR status, or the result (orresults or a summary of results) of the performed diagnostic analysiswith the corresponding patient’s name, medical record,symbolic/numerical identifier, or a combination thereof. Suchidentification can be based solely on detecting the presence of an LOHsignature or can be based at least in part on detecting the presence ofan LOH signature. For example, a laboratory technician or laboratoryprofessional can identify a patient having cancer cells that weredetected to have an LOH signature as having cancer cells potentiallydeficient in HDR based on a combination of a positive LOH signaturestatus and the results of other genetic and biochemical tests performedat the testing laboratory. In some embodiments, the patients aretreatment naïve patients.

In some cases, a laboratory technician or laboratory professional orgroup of laboratory technicians or laboratory professionals can identifya patient having cancer cells that were detected to have an LOHsignature as having cancer cells potentially containing a geneticmutation in one or more genes in the HDR pathway by associating thepositive LOH signature status, the potential presence of a geneticmutation in one or more genes in the HDR pathway, or the result (orresults or a summary of results) of the performed diagnostic analysiswith the corresponding patient’s name, medical record,symbolic/numerical identifier, or a combination thereof. Suchidentification can be based solely on detecting the presence of an LOHsignature or can be based at least in part on detecting the presence ofan LOH signature. For example, a laboratory technician or laboratoryprofessional can identify a patient having cancer cells that weredetected to have an LOH signature as having cancer cells potentiallycontaining a genetic mutation in one or more genes in the HDR pathwaybased on a combination of a positive LOH signature status and theresults of other genetic and biochemical tests performed at the testinglaboratory. In some embodiments, the patients are treatment naïvepatients.

In some cases, a laboratory technician or laboratory professional orgroup of laboratory technicians or laboratory professionals can identifya patient having cancer cells that were detected to have an LOHsignature as having cancer cells likely to respond to a particularcancer treatment regimen by associating the positive LOH signaturestatus, a potentially deficient HDR status, a potential presence of adeficient status in one or more genes in the HDR pathway, or the result(or results or a summary of results) of the performed diagnosticanalysis with the corresponding patient’s name, medical record,symbolic/numerical identifier, or a combination thereof. Suchidentification can be based solely on detecting the presence of an LOHsignature or can be based at least in part on detecting the presence ofan LOH signature. For example, a laboratory technician or laboratoryprofessional can identify a patient having cancer cells that weredetected to have an LOH signature as having cancer cells likely torespond to a particular cancer treatment regimen based on a combinationof a positive LOH signature status and the results of other genetic andbiochemical tests performed at the testing laboratory. In someembodiments, the patients are treatment naïve patients.

Once a laboratory technician or laboratory professional or group oflaboratory technicians or laboratory professionals detects the absenceof an LOH signature, the laboratory technician or laboratoryprofessional (or group) can identify the patient whose cancer cells weredetected as lacking an LOH signature as having cancer cells with anegative LOH signature status. For example, one or more laboratorytechnicians or laboratory professionals can identify a patient havingcancer cells that were detected to lack an LOH signature as havingcancer cells with a negative LOH signature status by associating thatnegative LOH signature status or the result (or results or a summary ofresults) of the performed diagnostic analysis with the correspondingpatient’s name, medical record, symbolic/numerical identifier, or acombination thereof. In some cases, a laboratory technician orlaboratory professional or group of laboratory technicians or laboratoryprofessionals can identify a patient having cancer cells that weredetected to lack an LOH signature as having cancer cells withpotentially intact HDR by associating the negative LOH signature status,the potentially intact HDR status, or the result (or results or asummary of results) of the performed diagnostic analysis with thecorresponding patient’s name, medical record, symbolic/numericalidentifier, or a combination thereof. In some embodiments, the patientsare treatment naïve patients.

In some cases, a laboratory technician or laboratory professional orgroup of laboratory technicians or laboratory professionals can identifya patient having cancer cells that were detected to lack an LOHsignature as having cancer cells with potentially intact genes of theHDR pathway by associating the negative LOH signature status, thepotential absence of genetic mutations in genes of the HDR pathway, orthe result (or results or a summary of results) of the performeddiagnostic analysis with the corresponding patient’s name, medicalrecord, symbolic/numerical identifier, or a combination thereof. In someembodiments, the patients are treatment naïve patients.

In some cases, a laboratory technician or laboratory professional orgroup of laboratory technicians or laboratory professionals can identifya patient having cancer cells that were detected to lack an LOHsignature as having cancer cells as less likely to respond to oneparticular treatment (e.g., a platinum-based chemotherapy drug such ascisplatin, carboplatin, oxalaplatin, or picoplatin, an anthracyclinesuch as epirubincin or doxorubicin, a topoisomerase I inhibitor such ascampothecin, topotecan, or irinotecan, a PARP inhibitor such asiniparib, olaparib, or velapirib, or radiation) and/or more likely torespond to a particular cancer treatment regimen (e.g., a cancertreatment regimen that includes the use of a cancer treatment agent notassociated with HDR) by associating the negative LOH signature status, apotentially intact HDR status, a potential absence of genetic mutationsin genes of the HDR pathway, or the result (or results or a summary ofresults) of the performed diagnostic analysis with the correspondingpatient’s name, medical record, symbolic/numerical identifier, or acombination thereof. In some embodiments, the patients are treatmentnaïve patients.

Once a laboratory technician or laboratory professional or group oflaboratory technicians or laboratory professionals detects the presenceof an increased number of LOH regions that cover the whole chromosome,the laboratory technician or laboratory professional (or group) canidentify the patient whose cancer cells were detected as having anincreased number of LOH regions that cover the whole chromosome aslikely having cancer cells with an intact BRCA1, BRCA2 and/or RAD51Cstatus, or intact HDR pathway. For example, one or more laboratorytechnicians or laboratory professionals can identify a patient havingcancer cells that were detected to have an increased number of LOHregions that cover the whole chromosome as likely having cancer cellswith an intact BRCA1 and BRCA2 status by associating the presence of anincreased number of LOH regions that cover the whole chromosome or theresult (or results or a summary of results) of the performed diagnosticanalysis with the corresponding patient’s name, medical record,symbolic/numerical identifier, or a combination thereof. In someembodiments, the patients are treatment naïve patients.

The results of any analyses according to the invention will often becommunicated to physicians, genetic counselors and/or patients (or otherinterested parties such as researchers) in a transmittable form that canbe communicated or transmitted to any of the above parties. Such a formcan vary and can be tangible or intangible. The results can be embodiedin descriptive statements, diagrams, photographs, charts, images or anyother visual forms. For example, graphs or diagrams showing genotype orLOH (or HRD status) information can be used in explaining the results.The statements and visual forms can be recorded on a tangible mediumsuch as papers, computer readable media such as floppy disks, compactdisks, flash memory, etc., or in an intangible medium, e.g., anelectronic medium in the form of email or website on internet orintranet. In addition, results can also be recorded in a sound form andtransmitted through any suitable medium, e.g., analog or digital cablelines, fiber optic cables, etc., via telephone, facsimile, wirelessmobile phone, internet phone and the like.

Thus, the information and data on a test result can be produced anywherein the world and transmitted to a different location. As an illustrativeexample, when an assay is conducted outside the United States, theinformation and data on a test result may be generated, cast in atransmittable form as described above, and then imported into the UnitedStates. Accordingly, the present invention also encompasses a method forproducing a transmittable form of information on an LOH signature for atleast one patient sample. The method comprises the steps of (1)determining an LOH signature according to methods of the presentinvention; and (2) embodying the result of the determining step in atransmittable form. The transmittable form is a product of such amethod.

Several embodiments of the invention described herein involve a step ofcorrelating an LOH signature according to the present invention (e.g.,the total number of LOH regions in at least one pair of humanchromosomes of said cancer cell that are longer than a first length butshorter than the length of the whole chromosome containing the LOHregion, wherein said at least one pair of human chromosomes is not ahuman X/Y sex chromosome pair, wherein said first length is about 1.5 ormore megabases) to a particular clinical feature (e.g., an increasedlikelihood of a deficiency in the BRCA1 or BRCA2 gene; an increasedlikelihood of HDR deficiency; an increased likelihood of response to atreatment regimen comprising a DNA damaging agent, an anthracycline, atopoisomerase I inhibitor, radiation, and/or a PARP inhibitor; etc.) ifthe number is greater than some reference (or optionally to anotherfeature if the number is less than some reference). Throughout thisdocument, wherever such an embodiment is described, another embodimentof the invention may involve, in addition to or instead of a correlatingstep, one or both of the following steps: (a) concluding that thepatient has the clinical feature based at least in part on the presenceor absence of the LOH signature; or (b) communicating that the patienthas the clinical feature based at least in part on the presence orabsence of the LOH signature.

By way of illustration, but not limitation, one embodiment described inthis document is a method of predicting a cancer patient’s response to acancer treatment regimen comprising a DNA damaging agent, ananthracycline, a topoisomerase I inhibitor, radiation, and/or a PARPinhibitor, said method comprising: (1) determining, in a cancer cellfrom said cancer patient, the number of LOH regions in at least one pairof human chromosomes of a cancer cell of said cancer patient that arelonger than a first length but shorter than the length of the wholechromosome containing the LOH region, wherein said at least one pair ofhuman chromosomes is not a human X/Y sex chromosome pair, wherein saidfirst length is about 1.5 or more megabases; and (2) correlating saidtotal number that is greater than a reference number with an increasedlikelihood that said cancer patient will respond to said cancertreatment regimen. According to the preceding paragraph, thisdescription of this embodiment is understood to include a description oftwo related embodiments, i.e., a method of predicting a cancer patient’sresponse to a cancer treatment regimen comprising a DNA damaging agent,an anthracycline, a topoisomerase I inhibitor, radiation, and/or a PARPinhibitor, said method comprising: (1) determining, in a cancer cellfrom said cancer patient, the number of LOH regions in at least one pairof human chromosomes of a cancer cell of said cancer patient that arelonger than a first length but shorter than the length of the wholechromosome containing the LOH region, wherein said at least one pair ofhuman chromosomes is not a human X/Y sex chromosome pair, wherein saidfirst length is about 1.5 or more megabases; and (2)(a) concluding thatsaid patient has an increased likelihood that said cancer patient willrespond to said cancer treatment regimen based at least in part on atotal number that is greater than a reference number; or (2)(b)communicating that said patient has an increased likelihood that saidcancer patient will respond to said cancer treatment regimen based atleast in part on a total number that is greater than a reference number.

In each embodiment described in this document involving correlating aparticular assay or analysis output (e.g., total number of LOH regionsgreater than a reference number, etc.) to some likelihood (e.g.,increased, not increased, decreased, etc.) of some clinical feature(e.g., response to a particular treatment, cancer-specific death, etc.),or additionally or alternatively concluding or communicating suchclinical feature based at least in part on such particular assay oranalysis output, such correlating, concluding or communicating maycomprise assigning a risk or likelihood of the clinical featureoccurring based at least in part on the particular assay or analysisoutput. In some embodiments, such risk is a percentage probability ofthe event or outcome occurring. In some embodiments, the patient isassigned to a risk group (e.g., low risk, intermediate risk, high risk,etc.). In some embodiments “low risk” is any percentage probabilitybelow 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In someembodiments “intermediate risk” is any percentage probability above 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% and below 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some embodiments“high risk” is any percentage probability above 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

As used herein, “communicating” a particular piece of information meansto make such information known to another person or transfer suchinformation to a thing (e.g., a computer). In some methods of theinvention, a patient’s prognosis or likelihood of response to aparticular treatment is communicated. In some embodiments, theinformation used to arrive at such a prognosis or response prediction(e.g., LOH signature according to the present invention, etc.) iscommunicated. This communication may be auditory (e.g., verbal), visual(e.g., written), electronic (e.g., data transferred from one computersystem to another), etc. In some embodiments, communicating a cancerclassification (e.g., prognosis, likelihood of response, appropriatetreatment, etc.) comprises generating a report that communicates thecancer classification. In some embodiments the report is a paper report,an auditory report, or an electronic record. In some embodiments thereport is displayed and/or stored on a computing device (e.g., handhelddevice, desktop computer, smart device, website, etc.). In someembodiments the cancer classification is communicated to a physician(e.g., a report communicating the classification is provided to thephysician). In some embodiments the cancer classification iscommunicated to a patient (e.g., a report communicating theclassification is provided to the patient). Communicating a cancerclassification can also be accomplished by transferring information(e.g., data) embodying the classification to a server computer andallowing an intermediary or end-user to access such information (e.g.,by viewing the information as displayed from the server, by downloadingthe information in the form of one or more files transferred from theserver to the intermediary or end-user’s device, etc.).

Wherever an embodiment of the invention comprises concluding some fact(e.g., a patient’s prognosis or a patient’s likelihood of response to aparticular treatment regimen), this may include in some embodiments acomputer program concluding such fact, typically after performing analgorithm that applies information on LOH regions according to thepresent invention.

In each embodiment described herein involving a number of LOH regions(e.g., LOH Indicator Regions) or a total combined length of such LOHregions, the present invention encompasses a related embodimentinvolving a test value or score (e.g., HRD score, LOH score, etc.)derived from, incorporating, and/or, at least to some degree, reflectingsuch number or length. In other words, the bare LOH region numbers orlengths need not be used in the various methods, systems, etc. of theinvention; a test value or score derived from such numbers or lengthsmay be used. For example, one embodiment of the invention provides amethod of treating cancer in a patient, comprising: (1) determining in asample from said patient the number of LOH regions in at least one pairof human chromosomes of a cancer cell of the cancer patient that arelonger than a first length but shorter than the length of the wholechromosome containing the LOH region indicates that the cancer cellshave the LOH signature, wherein the at least one pair of humanchromosomes is not a human X/Y sex chromosome pair, wherein the firstlength is about 1.5 or more megabases; (2) providing a test valuederived from the number of said LOH regions; (3) comparing said testvalue to one or more reference values derived from the number of saidLOH regions in a reference population (e.g., mean, median, terciles,quartiles, quintiles, etc.); and (4)(a) administering to said patient ananti-cancer drug, or recommending or prescribing or initiating atreatment regimen comprising chemotherapy and/or a synthetic lethalityagent based at least in part on said comparing step revealing that thetest value is greater (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or10-fold greater; at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 standarddeviations greater) than at least one said reference value; or (4)(b)recommending or prescribing or initiating a treatment regimen notcomprising chemotherapy and/or a synthetic lethality agent based atleast in part on said comparing step revealing that the test value isnot greater (e.g., not more than 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or10-fold greater; not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 standarddeviations greater) than at least one said reference value. Theinvention encompasses, mutatis mutandis, corresponding embodiments wherethe test value or score is used to determine the patient’s prognosis,the patient’s likelihood of response to a particular treatment regimen,the patient’s or patient’s sample’s likelihood of having a BRCA1, BRCA2,RAD51C or HDR deficiency, etc.

FIG. 15 shows an exemplary process by which a computing system (or acomputer program (e.g., software) containing computer-executableinstructions) can identify LOH loci or regions from genotype data asdescribed herein. If the observed ratio of the signals of two alleles, Aand B, is two to one, there are two possibilities. The first possibilityis that cancer cells have LOH with deletion of allele B in a sample with50% contamination with normal cells. The second possibility is thatthere is no LOH but allele A is duplicated in a sample with nocontamination with normal cells. The process begins at box 1500, wherethe following data are collected by the computing system; (1)sample-specific normalized signal intensities for both alleles of eachlocus and (2) assay-specific (specific for different SNP arrays and forsequence based approach) set of parameters defined based on analysis oflarge number of samples with known ASCN profiles. As described herein,any appropriate assay such as a SNP array-based assay orsequencing-based assay can be used to assess loci along a chromosome forhomozygosity or heterozygosity. In some cases, a system including asignal detector and a computer can be used to collect data (e.g.,fluorescent signals or sequencing results) regarding the homozygous orheterozygous nature of the plurality of loci (e.g., sample-specificnormalized signal intensities for both alleles of each locus). At box1510, allele specific copy numbers (ASCN) are reconstructed at eachlocus (e.g., each SNP). ASCNs are the numbers of copies of both paternaland maternal alleles. At box 1530, a likelihood function is used todetermine whether a homozygous locus or region of homozygous loci is dueto LOH. This can be conceptually analogous to a previously describedalgorithm designed to reconstruct total copy number (rather than ASCN)at each locus (e.g., SNP). See International Application No.PCT/US2011/026098 to Abkevich et al. The likelihood function can bemaximized over ASCN of all loci, level of contamination with benigntissue, total copy number averaged over the whole genome, and samplespecific noise level. At box 1540, an LOH region is determined as astretch of SNPs with one of the ASCNs (paternal or maternal) being zero.In some embodiments, the computer process further comprises a step ofinquiring or determining whether a patient is treatment naïve.

FIG. 3 shows an exemplary process by which a computing system candetermine the presence or absence of an LOH signature. The processbegins at box 300, where data regarding the homozygous or heterozygousnature of a plurality of loci along a chromosome is collected by thecomputing system. As described herein, any appropriate assay such as aSNP array-based assay or sequencing-based assay can be used to assessloci along a chromosome for homozygosity or heterozygosity. In somecases, a system including a signal detector and a computer can be usedto collect data (e.g., fluorescent signals or sequencing results)regarding the homozygous or heterozygous nature of the plurality ofloci. At box 310, data regarding the homozygous or heterozygous natureof a plurality of loci as well as the location or spatial relationshipof each locus is assessed by the computing system to determine thelength of any LOH regions present along a chromosome. At box 320, dataregarding the number of LOH regions detected and the length of eachdetected LOH region is assessed by the computing system to determine thenumber of LOH regions that have a length (a) greater than or equal to apreset number of Mb (e.g., 15 Mb) and (b) less than the entire length ofthe chromosome containing that LOH region. Alternatively the computingsystem can determine the total or combined LOH length as describedabove. At box 330, the computing system formats an output providing anindication of the presence or absence of an LOH signature. Onceformatted, the computing system can present the output to a user (e.g.,a laboratory technician, clinician, or medical professional). Asdescribed herein, the presence or absence of an LOH signature can beused to provide an indication about a patient’s likely HDR status, anindication about the likely presence or absence of genetic mutations ingenes of the HDR pathway, and/or an indication about possible cancertreatment regimens.

FIG. 4 is a diagram of an example of a computer device 1400 and a mobilecomputer device 1450, which may be used with the techniques describedherein. Computing device 1400 is intended to represent various forms ofdigital computers, such as laptops, desktops, workstations, personaldigital assistants, servers, blade servers, mainframes, and otherappropriate computers. Computing device 1450 is intended to representvarious forms of mobile devices, such as personal digital assistants,cellular telephones, smart phones, and other similar computing devices.The components shown here, their connections and relationships, andtheir functions, are meant to be exemplary only, and are not meant tolimit implementations of the inventions described and/or claimed in thisdocument.

Computing device 1400 includes a processor 1402, memory 1404, a storagedevice 1406, a high-speed interface 1408 connecting to memory 1404 andhigh-speed expansion ports 1410, and a low speed interface 1415connecting to low speed bus 1414 and storage device 1406. Each of thecomponents 1402, 1404, 1406, 1408, 1410, and 1415, are interconnectedusing various busses, and may be mounted on a common motherboard or inother manners as appropriate. The processor 1402 can processinstructions for execution within the computing device 1400, includinginstructions stored in the memory 1404 or on the storage device 1406 todisplay graphical information for a GUI on an external input/outputdevice, such as display 1416 coupled to high speed interface 1408. Inother implementations, multiple processors and/or multiple buses may beused, as appropriate, along with multiple memories and types of memory.Also, multiple computing devices 1400 may be connected, with each deviceproviding portions of the necessary operations (e.g., as a server bank,a group of blade servers, or a multi-processor system).

The memory 1404 stores information within the computing device 1400. Inone implementation, the memory 1404 is a volatile memory unit or units.In another implementation, the memory 1404 is a non-volatile memory unitor units. The memory 1404 may also be another form of computer-readablemedium, such as a magnetic or optical disk.

The storage device 1406 is capable of providing mass storage for thecomputing device 1400. In one implementation, the storage device 1406may be or contain a computer-readable medium, such as a floppy diskdevice, a hard disk device, an optical disk device, or a tape device, aflash memory or other similar solid state memory device, or an array ofdevices, including devices in a storage area network or otherconfigurations. A computer program product can be tangibly embodied inan information carrier. The computer program product may also containinstructions that, when executed, perform one or more methods, such asthose described herein. The information carrier is a computer- ormachine-readable medium, such as the memory 1404, the storage device1406, memory on processor 1402, or a propagated signal.

The high speed controller 1408 manages bandwidth-intensive operationsfor the computing device 1400, while the low speed controller 1415manages lower bandwidth-intensive operations. Such allocation offunctions is exemplary only. In one implementation, the high-speedcontroller 1408 is coupled to memory 1404, display 1416 (e.g., through agraphics processor or accelerator), and to high-speed expansion ports1410, which may accept various expansion cards (not shown). In theimplementation, low-speed controller 1415 is coupled to storage device1406 and low-speed expansion port 1414. The low-speed expansion port,which may include various communication ports (e.g., USB, Bluetooth,Ethernet, or wireless Ethernet) may be coupled to one or moreinput/output devices, such as a keyboard, a pointing device, a scanner,an optical reader, a fluorescent signal detector, or a networking devicesuch as a switch or router, e.g., through a network adapter.

The computing device 1400 may be implemented in a number of differentforms, as shown in the figure. For example, it may be implemented as astandard server 1420, or multiple times in a group of such servers. Itmay also be implemented as part of a rack server system 1424. Inaddition, it may be implemented in a personal computer such as a laptopcomputer 1422. Alternatively, components from computing device 1400 maybe combined with other components in a mobile device (not shown), suchas device 1450. Each of such devices may contain one or more ofcomputing device 1400, 1450, and an entire system may be made up ofmultiple computing devices 1400, 1450 communicating with each other.

Computing device 1450 includes a processor 1452, memory 1464, aninput/output device such as a display 1454, a communication interface1466, and a transceiver 1468, among other components (e.g., a scanner,an optical reader, a fluorescent signal detector). The device 1450 mayalso be provided with a storage device, such as a microdrive or otherdevice, to provide additional storage. Each of the components 1450,1452, 1464, 1454, 1466, and 1468, are interconnected using variousbuses, and several of the components may be mounted on a commonmotherboard or in other manners as appropriate.

The processor 1452 can execute instructions within the computing device1450, including instructions stored in the memory 1464. The processormay be implemented as a chipset of chips that include separate andmultiple analog and digital processors. The processor may provide, forexample, for coordination of the other components of the device 1450,such as control of user interfaces, applications run by device 1450, andwireless communication by device 1450.

Processor 1452 may communicate with a user through control interface1458 and display interface 1456 coupled to a display 1454. The display1454 may be, for example, a TFT LCD (Thin-Film-Transistor Liquid CrystalDisplay) or an OLED (Organic Light Emitting Diode) display, or otherappropriate display technology. The display interface 1456 may compriseappropriate circuitry for driving the display 1454 to present graphicaland other information to a user. The control interface 1458 may receivecommands from a user and convert them for submission to the processor1452. In addition, an external interface 1462 may be provide incommunication with processor 1452, so as to enable near areacommunication of device 1450 with other devices. External interface 1462may provide, for example, for wired communication in someimplementations, or for wireless communication in other implementations,and multiple interfaces may also be used.

The memory 1464 stores information within the computing device 1450. Thememory 1464 can be implemented as one or more of a computer-readablemedium or media, a volatile memory unit or units, or a non-volatilememory unit or units. Expansion memory 1474 may also be provided andconnected to device 1450 through expansion interface 1472, which mayinclude, for example, a SIMM (Single In Line Memory Module) cardinterface. Such expansion memory 1474 may provide extra storage spacefor device 1450, or may also store applications or other information fordevice 1450. For example, expansion memory 1474 may include instructionsto carry out or supplement the processes described herein, and mayinclude secure information also. Thus, for example, expansion memory1474 may be provide as a security module for device 1450, and may beprogrammed with instructions that permit secure use of device 1450. Inaddition, secure applications may be provided via the SIMM cards, alongwith additional information, such as placing identifying information onthe SIMM card in a non-hackable manner.

The memory may include, for example, flash memory and/or NVRAM memory,as discussed below. In one implementation, a computer program product istangibly embodied in an information carrier. The computer programproduct contains instructions that, when executed, perform one or moremethods, such as those described herein. The information carrier is acomputer- or machine-readable medium, such as the memory 1464, expansionmemory 1474, memory on processor 1452, or a propagated signal that maybe received, for example, over transceiver 1468 or external interface1462.

Device 1450 may communicate wirelessly through communication interface1466, which may include digital signal processing circuitry wherenecessary. Communication interface 1466 may provide for communicationsunder various modes or protocols, such as GSM voice calls, SMS, EMS, orMMS messaging, CDMA, TDMA, PDC, WCDMA, CDMA2000, or GPRS, among others.Such communication may occur, for example, through radio-frequencytransceiver 1468. In addition, short-range communication may occur, suchas using a Bluetooth, WiFi, or other such transceiver (not shown). Inaddition, GPS (Global Positioning System) receiver module 1470 mayprovide additional navigation- and location-related wireless data todevice 1450, which may be used as appropriate by applications running ondevice 1450.

Device 1450 may also communicate audibly using audio codec 1460, whichmay receive spoken information from a user and convert it to usabledigital information. Audio codec 1460 may likewise generate audiblesound for a user, such as through a speaker, e.g., in a handset ofdevice 1450. Such sound may include sound from voice telephone calls,may include recorded sound (e.g., voice messages, music files, etc.) andmay also include sound generated by applications operating on device1450.

The computing device 1450 may be implemented in a number of differentforms, as shown in the figure. For example, it may be implemented as acellular telephone 1480. It may also be implemented as part of asmartphone 1482, personal digital assistant, or other similar mobiledevice.

Various implementations of the systems and techniques described hereincan be realized in digital electronic circuitry, integrated circuitry,specially designed ASICs (application specific integrated circuits),computer hardware, firmware, software, and/or combinations thereof.These various implementations can include implementation in one or morecomputer programs that are executable and/or interpretable on aprogrammable system including at least one programmable processor, whichmay be special or general purpose, coupled to receive data andinstructions from, and to transmit data and instructions to, a storagesystem, at least one input device, and at least one output device.

These computer programs (also known as programs, software, softwareapplications or code) include machine instructions for a programmableprocessor, and can be implemented in a high-level procedural and/orobject-oriented programming language, and/or in assembly/machinelanguage. As used herein, the terms “machine-readable medium” and“computer-readable medium” refer to any computer program product,apparatus and/or device (e.g., magnetic discs, optical disks, memory,and Programmable Logic Devices (PLDs)) used to provide machineinstructions and/or data to a programmable processor, including amachine-readable medium that receives machine instructions as amachine-readable signal. The term “machine-readable signal” refers toany signal used to provide machine instructions and/or data to aprogrammable processor.

To provide for interaction with a user, the systems and techniquesdescribed herein can be implemented on a computer having a displaydevice (e.g., a CRT (cathode ray tube) or LCD (liquid crystal display)monitor) for displaying information to the user and a keyboard and apointing device (e.g., a mouse or a trackball) by which the user canprovide input to the computer. Other kinds of devices can be used toprovide for interaction with a user as well; for example, feedbackprovided to the user can be any form of sensory feedback (e.g., visualfeedback, auditory feedback, or tactile feedback); and input from theuser can be received in any form, including acoustic, speech, or tactileinput.

The systems and techniques described herein can be implemented in acomputing system that includes a back end component (e.g., as a dataserver), or that includes a middleware component (e.g., an applicationserver), or that includes a front end component (e.g., a client computerhaving a graphical user interface or a Web browser through which a usercan interact with an implementation of the systems and techniquesdescribed herein), or any combination of such back end, middleware, orfront end components. The components of the system can be interconnectedby any form or medium of digital data communication (e.g., acommunication network). Examples of communication networks include alocal area network (“LAN”), a wide area network (“WAN”), and theInternet.

The computing system can include clients and servers. A client andserver are generally remote from each other and typically interactthrough a communication network. The relationship of client and serverarises by virtue of computer programs running on the respectivecomputers and having a client-server relationship to each other.

In some cases, a computing system provided herein can be configured toinclude one or more sample analyzers. A sample analyzer can beconfigured to produce a plurality of signals about genomic DNA of atleast one pair of human chromosomes of a cancer cell. For example, asample analyzer can produce signals that are capable of beinginterpreted in a manner that identifies the homozygous or heterozygousnature of loci along a chromosome. In some cases, a sample analyzer canbe configured to carry out one or more steps of a SNP array-based assayor sequencing-based assay and can be configured to produce and/orcapture signals from such assays. In some cases, a computing systemprovided herein can be configured to include a computing device. In suchcases, the computing device can be configured to receive signals from asample analyzer. The computing device can include computer-executableinstructions or a computer program (e.g., software) containingcomputer-executable instructions for carrying out one or more of themethods or steps described herein. In some cases, suchcomputer-executable instructions can instruct a computing device toanalyze signals from a sample analyzer, from another computing device,from a SNP array-based assay, or from a sequencing-based assay. Theanalysis of such signals can be carried out to determine genotypes,homozygosity at certain loci, regions of homozygosity, the number of LOHregions, to determine the size of LOH regions, to determine the numberof LOH regions having a particular size or range of sizes, to determinewhether or not a sample is positive for an LOH signature, to determinethe number of Indicator LOH Regions in at least one pair of humanchromosomes, to determine a likelihood of a deficiency in BRCA1 and/orBRCA2 genes, to determine a likelihood of a deficiency in HDR, todetermine a likelihood that a cancer patient will respond to aparticular cancer treatment regimen (e.g., a regimen that includes a DNAdamaging agent, an anthracycline, a topoisomerase I inhibitor,radiation, a PARP inhibitor, or a combination thereof), or to determinea combination of these items.

In some cases, a computing system provided herein can includecomputer-executable instructions or a computer program (e.g., software)containing computer-executable instructions for formatting an outputproviding an indication about the number of LOH regions, the size of LOHregions, the number of LOH regions having a particular size or range ofsizes, whether or not a sample is positive for an LOH signature, thenumber of Indicator LOH Regions in at least one pair of humanchromosomes, a likelihood of a deficiency in BRCA1 and/or BRCA2 genes, alikelihood of a deficiency in HDR, a likelihood that a cancer patientwill respond to a particular cancer treatment regimen (e.g., a regimenthat includes a DNA damaging agent, an anthracycline, a topoisomerase Iinhibitor, radiation, a PARP inhibitor, or a combination thereof), or acombination of these items. In some cases, a computing system providedherein can include computer-executable instructions or a computerprogram (e.g., software) containing computer-executable instructions fordetermining a desired cancer treatment regimen for a particular patientbased at least in part on the presence or absence of an LOH signature oron the number of Indicator LOH Regions.

In some cases, a computing system provided herein can include apre-processing device configured to process a sample (e.g., cancercells) such that a SNP array-based assay or sequencing-based assay canbe performed. Examples of pre-processing devices include, withoutlimitation, devices configured to enrich cell populations for cancercells as opposed to non-cancer cells, devices configured to lyse cellsand/or extract genomic nucleic acid, and devices configured to enrich asample for particular genomic DNA fragments.

This document also provides kits for assessing samples (e.g., cancercells) as described herein. For example, this document provides kits forassessing cancer cells for the presence of an LOH signature or todetermine the number of Indicator LOH Regions in at least one pair ofhuman chromosomes. A kit provided herein can include either SNP probes(e.g., an array of SNP probes for carrying out a SNP array-based assaydescribed herein) or primers (e.g., primers designed for sequencing SNPregions via a sequencing-based assay) in combination with a computerprogram product containing computer-executable instructions for carryingout one or more of the methods or steps described herein (e.g.,computer-executable instructions for determining the number of LOHregions having a particular size or range of sizes). In some cases, akit provided herein can include at least 500, 1000, 10,000, 25,000, or50,000 SNP probes capable of hybridizing to polymorphic regions of humangenomic DNA. In some cases, a kit provided herein can include at least500, 1000, 10,000, 25,000, or 50,000 primers capable of sequencingpolymorphic regions of human genomic DNA. In some cases, a kit providedherein can include one or more other ingredients for performing a SNParray-based assay or a sequencing-based assay. Examples of such otheringredients include, without limitation, buffers, sequencingnucleotides, enzymes (e.g., polymerases), etc. This document alsoprovides the use of any appropriate number of the materials providedherein in the manufacture of a kit for carrying out one or more of themethods or steps described herein. For example, this document providesthe use of a collection of SNP probes (e.g., a collection of 10,000 to100,000 SNP probes) and a computer program product provided herein inthe manufacture of a kit for assessing cancer cells for the presence ofan LOH signature. As another example, this document provides the use ofa collection of primers (e.g., a collection of 10,000 to 100,000 primersfor sequencing SNP regions) and a computer program product providedherein in the manufacture of a kit for assessing cancer cells for thepresence of an LOH signature.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 - Assessing LOH Regions and HDR

Two sets of tumors were used from advanced ovarian cancer patients. Thefirst set of 94 tumors (training set) was used to derive a candidatesignature, and the second set of 40 tumors (validation set) was used tovalidate the signature. All coding regions of BRCA1 and BRCA2 genes weresequenced to detect germ line and somatic mutations. Levels of BRCA1 andBRCA2 mRNA expression were measured, and Affymetrix SNP microarrays wereperformed.

A computer program was used to reconstruct LOH signature status based onallele intensities derived from the microarray data. An algorithm wasdeveloped and implemented as a computer program to reconstruct LOHregions based on genotype (e.g., SNP genotype) data.

One point of the algorithm was to first reconstruct allele specific copynumbers (ASCN) at each locus (e.g., SNP). ASCNs are the numbers ofcopies of both paternal and maternal alleles. An LOH region was thendetermined as a stretch of SNPs with one of the ASCNs (paternal ormaternal) being zero. The algorithm was based on maximizing a likelihoodfunction and was conceptually analogous to a previously describedalgorithm designed to reconstruct total copy number (rather than ASCN)at each locus (e.g., SNP). See International Application No.PCT/US2011/026098 to Abkevich et al. The likelihood function wasmaximized over ASCN of all loci, level of contamination with benigntissue, total copy number averaged over the whole genome, and samplespecific noise level. The input data for the algorithm included (1)sample-specific normalized signal intensities for both allele of eachlocus and (2) assay-specific (specific for different SNP arrays and forsequence based approach) set of parameters defined based on analysis oflarge number of samples with known ASCN profiles.

Tumors were defined as being HDR deficient for the purpose of thisanalysis if they either had one or more deleterious mutations in BRCA1and/or BRCA2 genes or if they had low expression of BRCA1 mRNA. The restof the tumors were defined as likely HDR non-deficient for the purposeof this analysis.

The distribution of the lengths of LOH regions was investigated (FIG. 5). Three categories of LOH regions were used: (1) LOH affecting a wholechromosome; (2) large LOH regions (greater than about 15 Mb), whichtypically affect a part of a chromosomal arm or the whole chromosomalarm; and (3) multiple short LOH regions (less than about 15 Mb). Second,using the training set only, the number of LOH regions of one of thesethree categories was assessed for possible correlations with HDRdeficiency. It was discovered that (1) the number of short LOH regionsdid not significantly correlate with HDR deficiency (p>0.05); (2) LOHcovering an entire chromosome correlated weakly with HDR deficiency(p=0.0011); and (3) the number of large LOH regions correlatedsignificantly with HDR deficiency (p=1.9e-8). More specifically, it wasdiscovered that all HDR deficient tumors had a high number of large LOHregions (e.g., nine or more), while the majority of tumors likely to beHDR non-deficient had a small number of large LOH regions (FIGS. 6-8 ).It was probable that tumors likely to be HDR non-deficient were in factHDR deficient due to other genetic alterations, excluding BRCA1 andBRCA2 mutations and low mRNA expression. In addition to the number oflarge LOH regions, the total length of these regions also correlatedsignificantly with HDR deficiency.

These results were confirmed with the validation set: (1) the number ofshort LOH regions did not significantly correlate with HDR deficiency(p>0.05); (2) LOH covering an entire chromosome correlated weakly withHDR deficiency (p=0.05); and (3) the number of large LOH regionscorrelated significantly with HDR deficiency (p=3.9e-6).

The 134 tumors were divided from combined training and validation datasets into three groups: (1) BRCA deficient if they either had one ormore deleterious mutations in BRCA1 and/or BRCA2 genes or if they hadlow expression of BRCA1 mRNA; (2) HDR deficient / BRCA intact if theyhave 9 or more large LOH regions (greater than 15 Mb but less than thelength of the entire chromosome); (3) HDR intact if they have less than9 large LOH regions (greater than 15 Mb but less than the length of theentire chromosome). Results of this analysis are presented in FIG. 9 .It shows a high frequency of BRCA deficiency as well as HDR deficiencythat is not due to BRCA deficiency among ovarian tumors.

FIG. 10 shows the distribution of large LOH regions (greater than 15 Mbbut less than the length of the entire chromosome) for different typesof cancer cell lines. The size of the circles is proportional to thenumber of samples with such number of large LOH regions. Frequency ofHDR deficiency (cell lines with at least 9 of such large LOH regions) isthe highest among breast and esophagus cancer cell lines. No HDRdeficiency was observed among colon cancer cell lines. Validating theprevious findings for ovarian tumors, all BRCA deficient cell lines werefound to be HDR deficient as well.

FIG. 11 shows the distribution of large LOH regions (greater than 15 Mbbut less than the length of the entire chromosome) for publiclyavailable lung tumor data set (GSE19399 from Gene Expression Omnibus).It was observed that frequency of HDR deficiency (defined as having atleast 9 large LOH regions) is quite large among lung tumors (39%).

In FIG. 12 the results of analysis of different tumors and cell linesare summarized. Frequency of HDR deficiency defined as fraction ofsamples with at least 9 large LOH regions (greater than 15 Mb but lessthan the length of the entire chromosome) is presented for severaltumors and cell lines. This frequency is as high as 50% among ovariantumors and was not observed at all among brain and colon cell lines.Thus it appears that HDR deficiency plays an important role for themajority of cancers.

Example 2 - Chemo Toxicity Responses

In preparation of chemo toxicity response experiments, all cell lineswere grown at 37° C. plus 5% CO₂ in 75 cm² tissue culture flasks (VWRInternational, Inc. Cat # 353136) and the recommended growth medium.Before performing each experiment, each cell line was trypsinized(Invitrogen Corporation Cat # 25200-056), counted, and seeded inAdvanced RPMI 1640 (Invitrogen Corporation Cat # 12633-020), 3% FBS, 1%penicillin/streptomycin (Invitrogen Corporation Cat # 15140-122) at 2500cells or 5000 cells in 100 µL media per well from columns 2-12 of96-well polystyrene microplates with clear bottom (Perkin Elmer Cat#6005181), leaving column 1 with 100 µL per well of media only. Thecell-seeded plates were then incubated at 37° C. plus 5% CO₂ overnight.

Two different final drug concentration working stocks were prepared. Incases where 100% DMSO was required for drug solubility, Advanced RPMI1640 was used as the diluent for the highest concentration. AdvancedRPMI 1640 plus a predetermined amount of DMSO equal to the total DMSO inthe high concentration working stock was used for the low concentration,with a maximum of 60% DMSO used for the lowest concentration. This wasdone to keep the DMSO concentrations equal in every well and preventnon-specific cell death as a result of DMSO. The lower of the two drugconcentrations was placed in a 96-well, thin-wall PCR cycle plate(Robbins Scientific Cat # 1055-00-0) in rows A-D, column 12, while thehigher concentration was placed in rows E-H, column 12, of the sameplate. Serial dilutions of 1:2 or 1:3 were performed in a descendingmanner from column 12 to 3, leaving columns 1 and 2 to be used for nocell/no drug and no drug controls. This allowed for quadruplet datapoints for each drug concentration. Once dilutions were complete, 5 µLwas transferred from the dilution plate to the corresponding well of theseeded cell plate. Plates receiving drugs were then incubated at 37° C.plus 5% CO₂ for either 3 days or 6 days.

Following a 3-day or 6-day dose regimen, ATPlite assays (Perkin Elmercat # 6016941) were run on each well of each plate according to theATPLite Assay protocol. The luminescence was then read on a FUSIONmachine and saved as a .CSV file. For each cell-line and drugcombination, the four replicates of the no-drug control were averagedand divided by 100 to create a “normalization factor” used to calculatea normalized percent survival. The normalized percent survival for theno-drug controls was 100%. The four replicates of the cell-plus-drugwells were averaged and divided by the normalization factor for eachdrug concentration. The percent survival for each drug concentration,starting with a concentration equal to 0, was used to calculate an IC₅₀using proprietary software.

FIG. 13 shows response to chemotherapy for breast and ovarian cancercell lines. On y-axis are indicated values of Log₁₀(IC₅₀) for differentchemotherapy drugs (camptothecin, as well as averaged results forplatinum compounds (oxaliplatin, cisplatin, and carboplatin) oranthracyclines (doxorubicin and epirubicin)) when exposed to 29 breastcancer cell lines as well as Log₁₀(IC₅₀) of paclitaxel when exposed to27 ovarian cancer cell lines. On the x-axis the number of large LOHregions longer than 15 Mb and shorter than the entire chromosome areindicated for these cell lines. The dashed lines place a thresholdnumber at nine.

FIG. 14 is a version of a graph from FIG. 13 that indicates specificityand sensitivity among responders and non-responders to treatment withplatinum compounds (oxaliplatin, cisplatin, and carboplatin) whenexposed to 29 breast cancer cell lines. The dashed lines place athreshold number of large LOH regions longer than 15 Mb and shorter thanthe entire chromosome at nine. The solid line divides cell lines intoresponders and non-responders.

Example 3 - Further Validation of HR Deficiency Assay Materials andMethods Ovarian Tumor Samples

Three independent human ovarian cancer cohorts were used. 1: 152unselected ovarian cancer samples. 2: 53 high grade serous ovariantumors. 3: Publicly available data from 435 serous ovarian cancersamples for which complete information was available were downloadedfrom The Cancer Genome Atlas (TCGA) Network web site on Oct. 31, 2011.All cohorts were obtained under Institutional Review Board(IRB)-approved protocols. Patient and tumor characteristics are shown inTable 2. Varying numbers of samples were utilized in the assaysdescribed (Table 3).

TABLE 2 Patient and cancer characteristics. First cohort Second cohortThird cohort Total Number of Patients 152 53 435 Age at diagnosis Range37 - 88 38-77 30 - 89 Median 59 56 59 Unknown 4 (2.6%) 0 0 Follow-uptime Range 20-5570 213 - 3294 8 - 5480 Median 1127 701 874 Unknown 5(3.2%) 0 2 (0.46%) 1 9 (5.9%) 0 6 (1.38%) 2 14 (9.2%) 0 21 (4.83%) 3 107(70.4%) 46 (86.8%) 338 (77.70%) 4 21 (13.8%) 7 (13.2%) 69 (15.86%)Unknown 1 (0.7%) 0 1 (0.23%) Serous 133 (87.5%) 40 (75.5%) 435Non-serous 8 (5.3%) 4 (7.6%) (100.00%) Mixed 10 (6.6%) 1 (1.9%) 0Unknown 1 (0.7%) 8 (15.1%) 0 0 1 8 (5.3%) 1 (1.9%) 2 (0.46%) 2 18(11.8%) 12 (22.6%) 50 (11.49%) 3 126 (82.9%) 40 (75.5%) 373 (85.75%) 4 00 1 (0.23%) Unknown 0 0 8 (1.84%) 0 9 (5.9%) 0 84 (19.31%) <= 1 cm 95(62.5%) 44 (83%) 200 (45.98%) > 1 cm 40 (26.3%) 9 (17%) 102 (23.45%)Unknown 8 (5.3%) 0 49 (11.26%) Yes 152 (100%) 53 (100%) 386 (88.74%) No0 0 0 Unknown 0 0 49 (11.26%) Yes 52 (98.1%) 399 (91.72%) Platinum (cisor carboplatin)-based (no taxane) 139 (91.5%) 1 (1.9%) NA Platinum plusTaxane (paclitaxel or docetaxel)- 12 (7.9%) 51 (96.2%) NA based 128(83.6%) 0 23 (5.29%) No 7 (4.6%) 1 (1.9%) 13 (2.99%) Unknown 6 (4%)

TABLE 3 Number of samples used in each assay Cohort 1 Cohort 2 AssayNumber of samples Reason assay was not applied to all samples Number ofsamples Reason assay was not applied to all samples Affymetrix 500K SNParrays 152 not applicable 53 not applicable BRCA1 and BRCA2 tumorsequencing 150 sequencing failed 52 sequencing failed BRCA1 and BRCA2germline sequencing 19 normal tissue not available or no mutationdetected in tumor 11 normal tissue not available or no mutation detectedin tumor CCP and BRCA1 qPCR 137 insufficient tissue for RNA extraction53 not applicable BRCA1 and BRCA2 methylation analysis 126 insufficientDNA for analysis 34 insufficient DNA for analysis Other HR genemethylation analysis 92 insufficient DNA for analysis 0 insufficient DNAfor analysis

Cell Lines

67 cancer cell lines were analyzed (29 ovarian, 34 breast, 3 colon, 1pancreatic). Three breast cancer cell lines were obtained from DSMZ(Braunschweig, Germany). The colon, pancreatic, and remaining breastcancer cell lines were obtained from ATCC (Manassas, VA). Cancer celllines were grown in RPMI + 10% FBS + 1% penicillin/streptomycin media at37° C. in T75 flasks until ~5×10⁶ cell density. Exceptions were celllines that required non-standard media, L-glutamine, or insulin. Cellsgrown in suspension were centrifuged for 5 minutes at 1700 rpm in a 1.5mL centrifuge tube and the supernatant discarded. Cells grown in amonolayer had medium removed by aspiration, were washed with PBS, andtrypsin solution added. After the cells detached they were collected inmedium, transferred to a 1.5 mL microcentrifuge tube and centrifuged at1700 rpm for 5 minutes. The supernatant was discarded. Isolated cellswere resuspended in 200 µL PBS.

Extraction of Genomic DNA and Total RNA from Frozen Tumors and CellLines

10 µm frozen sections were cut and macrodissected. The tissue washomogenized (TissueRuptor (Qiagen)) after addition of QlAzol lysisreagent, following by RNA isolation using a Qiagen miRNAeasy Mini Kitper the manufacturers protocol. A QIAamp DNA Mini Kit (Qiagen) was usedto isolate DNA as per the manufacturer’s protocol with an overnightlysis incubation at 56° C. and RNase A treatment.

BRCA1 and BRCA2 Sequencing

BRCA1 and BRCA2 sequencing was performed as described in Hennessy etal., 2010. Mutations identified were only included in the analyses ifclassified as deleterious or suspected deleterious based on previouslydescribed criteria (Beaudet and Tsui, 1993).

Promoter Methylation qPCR Assays

The Methyl-Profiler DNA Methylation PCR Array System (SABiosciences) wasused to quantify methylation levels following the manufacturersrecommended protocol. DNA methylation-sensitive andmethylation-dependent restriction enzymes were used to selectivelydigest unmethylated or methylated genomic DNA, respectively. Post-digestDNA was quantified by real-time PCR using primers flanking the regionsof interest. The relative concentrations of differentially methylatedDNA are determined by comparing the amount of each digest with that of amock digest.

BRCA1 Promoter Methylation Sequencing Assay

50 - 300 ng of DNA was incubated for ~5 hours at 60° C. with briefelevations to 95° C. under acidic conditions in the presence ofbisulfite. After incubation, the reaction was bound to a spin column andwashed under basic conditions to remove bisulfite, converted DNA wasthen eluted in 15 µL. Lower case region of primers is specific to thegenomic region being amplified. Upper case region of primers correspondsto the 454 Titanium chemistry tails and a 4 bp barcode (last 4 basesbefore the region specific bases). By combining the forward and reverseprimers in multiple combinations, it is possible to multiplex up to 100samples in a single sequence reaction.

BRCA1 and Cell Cycle Progression Signature Expression Assays

RNA was treated with Amplification Grade Deoxyribonuclease I(Sigma-Aldrich Inc.) per manufacturer’s protocol with an extendedincubation time of 30 minutes. Reverse transcription was performed usinga High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems,Foster City, CA) per manufacturer’s instructions.

Replicate preamplifications were run independently using the TaqmanPreAmp Master Mix Kit (Applied Biosystems) protocol in a 5 µl reactionvolume. To preamplification replicates were run at 8 and 18 cyclesrespectively for cell cycle gene assays. Three preamplificationreplicates were run at 18 cycles only for BRCA1 assays. Thepost-amplification products were diluted 1:5 in low-EDTA Tris-EDTA (TE).Quantitative Polymerase Chain Reaction (qPCR) was then performed andassessed on Gene Expression M48 Dynamic Arrays (Fluidigm, South SanFrancisco, CA) per manufacturer’s protocol. The comparative cyclethreshold (C_(T)) method was used to calculate relative gene expression.C_(T)S from preamplification of different numbers of cycles werecentered by the average of the genes on the replicate that were incommon between all replicates. The resulting values were normalizedfirst by the average C_(T)S of the housekeeper genes then by the averageof the normalized C_(T)S of each assay on all samples from the firstcohort to yield ΔΔC_(T). CCP score and relative BRCA1 expression werecalculated as the average of the negative of the ΔΔC_(T)s of thecell-cycle genes and BRCA1 assays, respectively.

Identification of Samples with Loss of BRCA1 Expression

Samples in which CCP expression and BRCA1 expression are anti-correlatedwere defined as BRCA1 deficient. The threshold for identifying patientswith abnormal BRCA1 expression was defined using robust linearregression in a large set of ovarian cancer samples (n = 300). BRCA1expression was regressed on CCP score using iteratively re-weightedleast squares (IWLS). Points outside of the 99% prediction interval onthe low end were considered abnormal. This method is described ingreater detail in International Application No. PCT/US2011/054369 toTimms et al.

Affymetrix 500 K GeneChip Arrays

The Affymetrix GeneChip Mapping Nspl or Styl Assay Kit was used in thegeneration of biotinylated DNA for Affymetrix Mapping 500K Nspl or Stylmicroarray hybridizations (each assay was prepared separately). GenomicDNA (250 ng) was digested with Nspl or Styl restriction enzyme andadaptors were added to restriction fragment ends with T4 DNA ligase.Adaptor-modified samples were PCR amplified using Clontech Titanium Taq,which generated an amplified product of average size between 200 and1,100 bp. Amplification products were purified using a Clontech DNAamplification cleanup kit. 90 µg of purified DNA was fragmented usingAffymetrix Fragmentation Reagent. Biotin-labeling of the fragmentedsample was accomplished using the GeneChip DNA Labeling Reagent.Biotin-labeled DNA was hybridized on Nspl or Styl Affymetrix microarraysat 49oC for 16 to 18 hours in the Affymetrix rotation oven. Afterhybridization, probe array wash and stain procedures were carried out onthe automatic Affymetrix Fluidics Stations as per manufacturer’s manualand microarrays were scanned and raw data was collected by AffymetrixGeneChip Scanner 3000.

CN and LOH analysis of SNP Microarray Data

The algorithm is designed to determine the most likely allele specificcopy number at each SNP location. The corresponding likelihoodexplicitly takes into account contamination of a cancer DNA sample withnon-cancer stromal cell DNA. A similar algorithm for CN analysis isdescribed in detail in International Application No. PCT/US2011/026098to Abkevich et al. (publication no. WO/2011/106541). The algorithm usedin this paper was implemented in two versions, one for analysis ofAffymetrix 500K GeneChip array data generated internally, and the otherfor analysis of GenomeWideSNP6 Affymetrix array data downloaded from theTCGA web site(http://tcga-data.nci.nih.gov/tcga/dataAccessMatrix.htm?diseaseType=OV).The latter array, in addition to SNP probes, contains a number of probesfor non-polymorphic locations across the human genome. These probes areinformative for CN analysis but are not directly informative for LOHanalysis.

Statistical Analysis

p-values in this paper were calculated using Kolmogorov-Smirnov testunless otherwise specified.

Results HR Deficient Tumors

A tumor sample was considered HR deficient if it had a germline orsomatic mutation in BRCA1 or BRCA2, or methylation or low mRNAexpression of BRCA1. 31 of 152 samples from the first cohort werecarriers of mutations in BRCA1 and/or BRCA2, along with 14/53 from thesecond cohort and 83/435 from the third cohort (two of which wereexcluded from the further analysis, see below). Mutations are summarizedin Table 4.

TABLE 4 BRCA1, BRCA2, and RAD51C defects detected in the study cohorts.Cohort N BRCA1 + BRCA2 mutation BRCA1 mutation BRCA2 mutation Total NRAD51C methylation RAD51C methylation + BRCA1 mutation 1 152 1 23 8 3289 2 1 2 53 0 11 3 14 ND ND ND 3 435 0 51 34¹ 85¹ 435 11 0 ¹ - Two ofthese mutations were excluded from the analysis because one copy ofBRCA2 remained intact.

The degree of methylation was measured for promoter CpG islands of bothBRCA1 and BRCA2. Methylation in multiple samples was observed for BRCA1,but not BRCA2. 11 of 126 samples from the first cohort, 3 of 34 from thesecond cohort and 64 of 435 from the third cohort were defined as HRdeficient due to high levels of BRCA1 promoter methylation. DeleteriousBRCA1 or BRCA2 mutations were not observed in any of these samples,except for one sample from the third cohort.

Low mRNA expression of BRCA1 or BRCA2 might also lead to HR deficiency,and be the result of mechanisms other than promoter methylation. BRCA1and BRCA2 expression levels were measured for 137 samples from the firstcohort and 53 samples from the second cohort. Expression of BRCA1 in 20samples was abnormally low. Only five samples with abnormally lowexpression of BRCA1 were not flagged as HR deficient due to BRCA1promoter methylation. No abnormally low expression was observed forBRCA2.

A single intact copy of BRCA1 or BRCA2 is required for functionality.For all BRCA1 deficient samples, the BRCA1 gene is contained within aregion of LOH. In addition, for all but two BRCA2 deficient samples, theBRCA2 gene is observed within an LOH region. These two BRCA2 deficientsamples were not considered HR deficient in our analysis.

Distribution of Lengths of LOH Regions

The initial hypothesis was that regions with LOH of different lengthmight appear in the cancer genome through different pathways, thusassociation between LOH and HR deficiency might depend on the length ofLOH regions. The distribution of lengths of LOH regions adjusted on thelength of chromosome arm on which these LOH regions have been observedis shown in FIG. 16 . Chromosomes 13, 14, 15, and 22 were excludedbecause SNPs are not available for the p arms of these chromosomes.Three distinct features were observed in this distribution. First, thereare many short LOH regions. Second, there is a long flat tail of LOHregions up to the length of a single chromosome arm. Few LOH regionscover more than one chromosome arm but less than the whole chromosome.Finally, there is a high peak corresponding to LOH over the wholechromosome. The observed distribution is quite different from thesimilar distribution obtained for CN variations (Beroukhim et al. 2010),this suggests that CN variations and LOH regions might arise viadifferent mechanisms.

Correlation Between Samples with HR Deficiency and LOH

The first cohort of samples was used as the “discovery” cohort. LOHregions on chromosome 17 were excluded from the analysis because inalmost all samples LOH was observed over this chromosome, probablybecause genes important for progression of ovarian cancer are on thischromosome. We checked for correlation between HR deficiency and thenumber of short LOH regions (<15 Mb), the number of long LOH regions(>15 Mb but less than the whole chromosome), and the number of LOHregions covering whole chromosomes. Various different LOH region lengthcut-offs can be used and the influence of this cut-off on detecting HRdeficiency is explored in FIG. 19 and its accompanying discussion,though 15 Mb was found to be generally preferred. There was nosignificant correlation between the number of short LOH regions and HRdeficiency. The number of LOH regions covering the whole chromosome wassignificantly larger in tumors with intact BRCA1 or BRCA2 (p=4×10⁻⁵).The number of long LOH regions (termed hereafter in this Example 3 andthroughout this document as “HRD score”) was significantly higher intumors with deficient BRCA1 or BRCA2 (p=9×10⁻¹¹) (FIG. 17 a ).

The second and third cohorts were used to validate the results obtainedfor the first cohort. The correlation between HR deficiency and numberof LOH regions covering whole chromosomes did not validate in the secondcohort, possibly due to low sample number, but was significantly larger(p=3×10⁻¹¹) among tumors with intact BRCA1 and BRCA2 in the thirdcohort. A highly significant correlation was observed between HRD scoreand HR deficiency for both cohorts (p=2×10⁻⁷ and p=9×10⁻³⁰ respectively)with HRD score being distinctly reduced among ovarian tumors with intactBRCA1 and BRCA2 (FIGS. 17 b and 17 c ).

Alterations in RAD51C and Other HR Pathway Genes

Available data suggest that BRCA1 and BRCA2 are the primary genesresponsible for HR deficiency in ovarian cancer. However, many othergenes may also be important with, for example, both RAD51C (Meindl etal., 2010) and RAD51D (Loveday et al., 2011) recently being implicatedas predisposition genes for ovarian cancer. The degree of methylationwas measured for promoter CpG islands of eight additional genes involvedin the HR pathway (Table 5) in the first cohort. Only RAD51C had highlevels of promoter methylation (3 of 89 samples). In the third cohort 11of 435 samples had methylation of the RAD51C promoter. All samplespositive for RAD51C methylation from both cohorts were homozygous at theRAD51C locus due to LOH. To test whether the HRD score is elevated insamples with RAD51C promoter methylation these samples from both cohortswere compared with BRCA intact samples without RAD51C methylation.Consistent with our observations for BRCA1 and BRCA2 genes, HRD scorewas significantly higher (p=0.0003) among samples with RAD51Cmethylation.

TABLE 5 Promoter methylation assays used (SABiosciences). Gene SymbolDescription Assay catalog ID MDC1 Mediator or DNA damage checkpoint 1MePH08721-2A PARP1 Poly(ADP-ribose) polymerase 1 MePH02379-2A BRCA1Breast Cancer 1, early onset MePH28472-1A BRCA2 Breast Cancer 2, earlyonset MePH28473-1A RAD50 RAD50 homolog MePH28350-1A RAD51C RAD51 homologC MePH22389-1A PALB2 Partner and localizer of BRCA2 MePH28516-1A CHEK2CHK2 checkpoint homolog MePH28264-1A ATM Ataxia telangiectasia mutatedMePH28470-1A RAD51 RAD51 homolog MePH19071-2A

In the third cohort deleterious mutations and methylation of HR pathwaygenes have been reported (TCGA, 2011). The mutations were examined andanalysis limited to defects with a high likelihood of being deleterious(e.g., nonsense and frameshift mutations), resulting in a total of 8deleterious mutations in 6 genes (ATM, ATR, FANCA, FANCD2, FANCM, andPALB2). An additional 5 samples had methylation of HR pathway genes.Loss of the second allele was detected in only 1 of the 13 samples (aFANCM nonsense mutation). Since deactivation of both alleles is neededto loose function of a tumor suppressor, most of these 13 samples areexpected to have intact HR. Not surprisingly, HRD score was not elevatedin the majority of these samples.

Analysis of Combined Data

Correlation between HRD score and HR deficiency (defined as deficiencyof BRCA1, BRCA2, or RAD51C) for all three cohorts is presented in theFIG. 17 d . A highly significant association is seen (p=2×10⁻⁵⁴).

An important question is whether the distribution of HRD scores is thesame for HR deficiency due to different genomic loci. To answer this,the distributions of HRD scores for BRCA1, BRCA2, and RAD51C deficienttumors were analyzed separately (FIG. 21 ). A significant difference wasobserved (p=7×10⁻⁵) with BRCA1 deficient samples having higher averageHRD score (16.1; SD=4.3) than BRCA2 deficient samples (13.0; SD=3.9).The differences in HRD scores between either BRCA1 or BRCA2 and RAD51C(14.5; SD=5.1) were not significant.

Normal tissue was available from some samples from the first two cohortsand all samples from the third cohort, this was used to determinewhether mutations in BRCA1 and BRCA2 were germline or somatic. There isno significant difference for somatic vs. germline in the distributionsof HRD scores for either BRCA1 or BRCA2 deficiency (FIG. 20 ).

HRD Score in BRCA1 and BRCA2 Deficient Cell Lines

Unselected breast (n=34) and ovarian (n=29) cell lines were obtainedfrom multiple sources; in addition 3 colon and one pancreatic cell linefrom NCl60 with published BRCA1 and BRCA2 status were analyzed. Of these67 cell lines, seven either carried homozygous deleterious mutations orhad methylation of the BRCA1 promoter, two had homozygous mutations withapparent functional reversion, and six carried heterozygous mutations.FIG. 18 a shows the distributions of HRD scores for these three groupsof mutants, as well as for wild type samples. The distributions of HRDscores among wild type ovarian tumors and wild type cancer cell linesare not significantly different. The distribution of HRD scores amongcancer cell lines with heterozygous mutations is similar to wild typecancer cell lines, presumably because cells become HR deficient onlywhen both copies of BRCA1 or BRCA2 are non-functional. For cancer celllines with functional loss of both copies of either BRCA1 or BRCA2,higher HRD scores are observed, similar to HRD scores observed forovarian tumors with BRCA1, BRCA2, or RAD51C deficient genes. HRD scoresare also high for cancer cell lines with reversion of BRCA1 and BRCA2mutations. This supports the original hypothesis that HR deficiencyresults in irreversible changes in LOH. The difference of thedistribution of HRD scores in either wild type or heterozygous mutantcell lines, and the distribution of HRD scores in cell lines with eitherhomozygous mutations (with or without reversion) or methylation of theBRCA1 promoter is highly significant (p=10⁻⁵). Importantly, there issignificant correlation between HRD score and BRCA1 and BRCA2 deficiencyafter excluding ovarian cancer cell lines from the dataset (p=0.01),suggesting that association of HRD score with HR deficiency is notrestricted to ovarian cancer.

Correlation Between HR Deficiency And Overall Survival (OS) AndProgression Free Survival (Pfs)

A significant correlation was observed between PFS (p=0.03) and OS(p=6×10⁻ ⁵) for the third cohort with improved survival for patientswith higher HRD scores (FIG. 18 b ). P-values were calculated using Coxmodel. The results are in agreement with, and extend previously reporteddata showing that germline mutations in BRCA1 and BRCA2 are associatedwith improved outcomes for ovarian cancer (Rubin et al., 1996, Boyd etal., 2000; Cass et al., 2003; Tan et al., 2008, Hennessy et al., 2010,).

Discussion

The HRD score was validated in two independent ovarian cancer datasets,and also reflected mutations resulting in HR deficiency in breast andpancreatic cell lines.

TABLE 6 Average of HRD score for BRCA1 and BRCA2 deficient and intacttumors and corresponding p values HR deficient (BRCA1 and BRCA2) HRintact (BRCA1 and BRCA2) HR deficient (BRCA1, BRCA2, and RAD51C) HRintact (BRCA1, BRCA2, and RAD51C) First cohort 15.9 (SD=4.6) 8.3(SD=6.1) 16.2 (SD=4.9) 8.0 (SD=5.8) p=9×10⁻¹¹ p=7×10⁻¹² Second cohort15.6 (SD=4.4) 5.6 (SD=4.9) 15.6 (SD=4.4) 5.6 (SD=4.9) p=2×10⁻⁷ p=2×10⁻⁷Third cohort 15.3 (SD=4.3) 8.8 (SD=5.0) 15.1 (SD=4.3) 8.6 (SD=5.0)p=9×10⁻³⁰ p=2×10⁻³² Combined data for three cohorts 15.5 (SD=4.4) 8.4(SD=5.3) 15.4 (SD=4.4) 8.2 (SD=5.2) p=10⁻⁴⁵ p=2×10⁻⁵⁴ Cancer cell lines19.7 (SD=4.6) 8.2 (SD=5.4) 19.7 (SD=4.6) 8.2 (SD=5.4) p=10⁻⁵ p=10⁻⁵

An intermediate class of LOH sizes greater than 15 Mb but less than awhole chromosome is highly positively correlated with defective HR genessuggesting that most if not all, of this type of LOH class existsbecause it incorporates double strand DNA breaks as part of its genesisand requires repair by HR. In contrast, LOH at the whole chromosomelevel is significantly less frequent in HR deficient tumors. Onepossible explanation is that LOH at the whole chromosome leveloriginates through an alternative competing mechanism that does notinvolve double strand DNA breaks.

In addition to BRCA1 and BRCA2 defects, RAD51C promoter methylation isobserved in ovarian tumors. High HRD score was significantly associatedwith RAD51C deficiency in two datasets. Only one additional HR deficienttumor was confirmed in the 3 datasets, a nonsense mutation in FANCM withLOH resulting in loss of the second allele. The HRD score associatedwith the FANCM mutation (8) is within the range of the normaldistribution for samples with elevated HRD score.

Among tumors with apparently intact BRCA1, BRCA2, and RAD51C, asubstantial fraction of the samples have an elevated HRD score. Onepossible explanation is that there is a substantial rate of defects inother genes in the HR pathway in many of these samples. An alternativeexplanation is that contamination of the tumor with normal tissuecomplicates detection of defects. Data suggest that the HRD score isless sensitive to contamination than other assays, and that undetecteddefects may explain a significant fraction of those samples withelevated HRD score (see Supplementary Results).

Published studies have demonstrated that secondary reversion mutationswhich restore BRCA2 function can arise in BRCA2 mutant cell lines afterexposure to platinum agents (Sakai et al., 2009; Sakai et al., 2008;Edwards et al., 2008). Norquist et al., (2011) observed thatapproximately 28% of recurrent tumors had a secondary mutation thatrestored BRCA function. Reversion mutations were seen primarily inindividuals with prior exposure to platinum agents and were predictiveof resistance to platinum. The HRD score results from cumulative defectsoccurring in the genome of the tumor. DNA based markers of HR deficiencyare likely to be strongly associated with HR deficiency because they arefunctionally linked to it. Consequently, the HRD score is a very robustmeasure of HR deficiency. However, its permanence means the score wouldlikely not be sensitive to reversion mutations. Post-treatment sampleswere not available from the tumors used in this study, however dataobtained from cell lines is consistent with this hypothesis. Failure todetect reversion mutations will result in false positives. This islikely to affect very few tumors in the neoadjuvant or adjuvant setting(Norquist et al., 2011) and is less of a concern than false negativeswhich would incorrectly identify individuals as likely non-responders.

High HRD score is highly correlated with HR deficiency, and this scorecan be utilized to identify patients with high likelihood of respondingto DNA damaging agents and PARP inhibitors (among other agents). Such atest has clear clinical utility in breast and ovarian cancer, and can beused to expand the use of PARPi and platinum salts to other cancerswhere HR deficiency is less well characterized.

Example 4 - Further Validation of HR Deficiency Assay Materials andMethods

The patient cohort analyzed in this example included 56 breast cancerpatients, all of whom are either BRCA mutation positive or have triplenegative breast cancer (most are TNBC). Stages I - III were included(most are II or III). The patients received 6 cycles of neoadjuvantgemcitabine + iniparib + carboplatin. Response was measured asrelatively lower residual cancer burden following treatment.

56 fresh frozen breast tumors were analyzed. Median degree ofcontamination is 60%. Nine samples had contamination of at least 90%. 11of these tumors were carriers of BRCA1 deleterious mutations and threewere carriers of BRCA2 deleterious mutations. In all of these tumorsthere was LOH at the deficient genes. One of the carriers of BRCA1deleterious mutations also carried a deleterious mutation in BRCA2.However in that sample there was no LOH at BRCA2 gene.

30 samples were obtained from patients who responded to treatment(residual cancer burden either 0 or 1). 13 of them are BRCA1/2deficient. 26 samples were obtained from non-responders (residual cancerburden either 2 or 3). One of them is BRCA1 deficient. Genotypinganalysis was performed by Affymetrix using Affymetrix MIP arrays (asdescribed in U.S. Pat. No. 6,858,412; U.S. Pat. Application PublicationNo. US20060234264; Hardenbol et al., Nature Biotechnology (2003) 21:673-678; Wang et al., BMC Med Genomics (2009) 2:8; each of which ishereby incorporated by reference in its entirety). HRD scores werecalculated as described above.

Results

The average HRD score for responders was 16.5. The average HRD score forBRCA1/2 intact and for BRCA1/2 deficient responders was the same. Theaverage HRD score for non-responders was 11.4. The average HRD score forBRCA1/2 intact non-responders is 11.6 and for BRCA1 deficientnon-responder was 8. According to the Mann-Whitney U test p-value forassociation between response to treatment and HRD score was 0.004. IfBRCA1/2 deficient samples are excluded association between response totreatment and HRD score remains significant (p-value = 0.02).

The differences in HRD score amongst samples with residual cancer burden0 and 1 were not significant. Similarly, the differences in HRD scoreamongst samples with residual cancer burden 2 and 3 were notsignificant. Correlations between response to treatment and clinicalparameters (stage, grade) were not significant.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-23. (canceled)
 24. A method, comprising: (i) genotyping a plurality ofsingle nucleotide polymorphism loci in at least five pairs of humanchromosomes in DNA from a cancer cell, (ii) detecting either ahomozygous or heterozygous genotype at each locus in the plurality ofsingle nucleotide polymorphism loci, wherein the plurality of singlenucleotide polymorphism loci comprises at least 10,000 single nucleotidepolymorphism loci, and (iii) detecting a homologous recombinationdeficiency in the cancer cell if a test value exceeds a reference value;or detecting no homologous recombination deficiency in the cancer cellif the test value does not exceed the reference value, wherein the testvalue is equal to or derived from the number of Indicator LOH Regions inthe at least five pairs of human chromosomes, and wherein the referencevalue is equal to or derived from a reference number of Indicator LOHRegions in at least five pairs of human chromosomes in cancer cellsamples of a population of reference patients.
 25. The method of claim24, wherein the number of Indicator LOH Regions is determined in atleast 10 pairs of human chromosomes.
 26. The method of claim 24, whereinthe number of Indicator LOH Regions is determined in at least 21 pairsof human chromosomes.
 27. The method of claim 24, wherein an IndicatorLOH Region is equal to or longer than 5 megabases.
 28. The method ofclaim 24, wherein an Indicator LOH Region is equal to or longer than 15megabases.
 29. The method of claim 24, wherein the reference number ofIndicator LOH Regions is at least nine.
 30. The method of claim 24,wherein the reference number of Indicator LOH Regions is at least 15.31. The method of claim 24, wherein the cancer cell is formalin-fixed,paraffin embedded (FFPE).
 32. The method of claim 24, wherein theplurality of single nucleotide polymorphism loci comprises at least25,000 single nucleotide polymorphism loci.
 33. The method of claim 24,wherein the plurality of single nucleotide polymorphism loci comprisesat least 50,000 single nucleotide polymorphism loci.
 34. The method ofclaim 24, wherein the cancer cell is a breast cancer cell or an ovariancancer cell.
 35. A method, comprising: (1) genotyping a plurality ofsingle nucleotide polymorphism loci in at least five pairs of humanchromosomes in DNA from a breast or ovarian cancer cell by (i) enrichingthe sample for DNA molecules each comprising at least one locus from theplurality of single nucleotide polymorphism loci, wherein there is atleast one single nucleotide polymorphism locus located on average every500 kb within each chromosome of the five pairs of human chromosomes and(ii) detecting either a homozygous or heterozygous genotype at eachlocus in the plurality of single nucleotide polymorphism loci, whereinthe plurality of single nucleotide polymorphism loci comprises at least1,000 single nucleotide polymorphism loci,, wherein an Indicator LOHRegion is defined as a genomic region wherein all single nucleotidepolymorphism the genotyped loci are homozygous, and wherein such genomicregion is equal to or longer than a first length but shorter than thelength of the whole chromosome containing the genomic region, andwherein the first length is at least 1.5 megabases; (2) providing a testvalue equal to or derived from the test number of Indicator LOH Regionsdetected in (1); (3) detecting a homologous recombination deficiency inthe cancer cell if said test value does not meet or exceed a referencevalue equal to or derived from a reference number of Indicator LOHRegions in at least ten pairs of human chromosomes in patient cancercell samples of a population of reference breast or ovarian cancerpatients, wherein said reference number of Indicator LOH Regions is atleast five.
 36. The method of claim 35, wherein the test number ofIndicator LOH Regions is determined in at least 10 pairs of humanchromosomes.
 37. The method of claim 35, wherein the length of the firstlength is at least 5 megabases.
 38. The method claim 35, wherein thelength of the first length is at least 15 megabases.
 39. The methodclaim 35, wherein the reference number of Indicator LOH Regions is atleast
 15. 40. The method of claim 35, wherein the plurality of singlenucleotide polymorphism loci comprises at least 2,500 single nucleotidepolymorphism loci.
 41. The method of claim 35, wherein the plurality ofsingle nucleotide polymorphism loci comprises at least 10,000 singlenucleotide polymorphism loci.
 42. The method of claim 35, wherein theplurality of single nucleotide polymorphism loci comprises at least25,000 single nucleotide polymorphism loci.
 43. The method of claim 35,wherein the plurality of single nucleotide polymorphism loci comprisesat least 50,000 single nucleotide polymorphism loci.